Scrub typhus: vector competence of Leptotrombidium chiangraiensis chiggers and transmission efficacy and isolation of Orientia tsutsugamushi.

A total of 4,281 rodents, belonging to 20 species, was collected from three villages in Chiangrai Province, northern Thailand, from January 1994 to December 2001. The predominant species were Rattus rattus, R. losea and Bandicota indica, accounting for 96% of the total. More than 135,000 chigger mites were removed from wild rodents of which 1% were identified as Leptotrombidium chiangraiensis, a new species vector of scrub typhus. Five Orientia tsutsugamushi-infected L. chiangraiensis colonies established from chiggers removed from R. rattus and R. losea rodents. The prevalence of O. tsutsugamushi infection in colonies ranged from 7-89%. Vertical and horizontal transmission efficacies of O. tsutsugamushi were stable between generations among colonies of L. chiangraiensis. The two isolates of O. tsutsugamushi obtained from individual adult L. chiangraiensis mites represent the first successful isolation of this bacterium from individual adult mites. Characterization of the O. tsutsugamushi isolates is under way.

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.

Investigation of the role of Blankaartia acuscutellaris (Acari: Trombiculidae) as a vector of scrub typhus in central Thailand.

Monthly collections of rodents were conducted in Phitsanulok Province, central Thailand in 1993 to investigate the role of the mite Blankaartia acuscurellaris as a vector of scrub typhus. Overall, a total of 41 rodents were collected and examined for the presence of the red colored larvae of B. acuscutellaris and yellow larvae of Leptotrombidium deliense and Ascoshoengastia sp. A total of 787 B. acuscutellaris and 1390 yellow larvae were placed into pools, triturated and isolation of Orientia tsutsugamushi attempted in laboratory mice. The sera of 8 of the collected rodents had elevated antibodies to O. tsutsugamushi indicating active infections; however, O. tsutsugamushi was not isolated from rodent tissues or pools of larvae. The results of this survey suggest that B. acuscutellaris may not be an important vector of scrub typhus, but more studies are needed in endemic areas.

Development and persistence of antibodies to Orientia tsutsugamushi in the roof rat, Rattus rattus and laboratory mice following attachment of naturally infected Leptotrombidium deliense.

The development and persistence of antibodies to Orientia tsutsugamushi in Rattus rattus and laboratory mice following infection from the bite of naturally infected Leptotrombidium deliense is reported. Antibodies in R. rattus were first detected using an indirect immunoperoxidase method 2 weeks after attachment of an infected mite. The IgM antibody response was mild, and was detected 2, 4 and 6 weeks after infection, while the IgG response was stronger and was detected from 2 to 19 weeks after infection, when the tests ceased. Active rickettsiae were isolated from R. rattus 1-8 weeks after attachment of infected L. deliense, but rats were not adversely affected by infection and appeared to behave in the same way as uninfected rats. In contrast to rats, laboratory mice were killed by O. tsutsugamushi following infection by attachment of infected L. deliense. The development of antibodies in mice was not detected before gross symptoms of infection were observed. Antibodies were first detected 14 days after attachment of infected mites, when mice were close to death.

Doxycycline and rifampicin for mild scrub-typhus infections in northern Thailand: a randomised trial.

BACKGROUND: Some strains of scrub typhus in northern Thailand are poorly responsive to standard antirickettsial drugs. We therefore did a masked, randomised trial to compare rifampicin with standard doxycycline therapy for patients with scrub typhus. METHODS: Adult patients with strictly defined, mild scrub typhus were initially randomly assigned 1 week of daily oral treatment with 200 mg doxycycline (n=40), 600 mg rifampicin (n=38), or doxycycline with rifampicin (n=11). During the first year of treatment, the combined regimen was withdrawn because of lack of efficacy and the regimen was replaced with 900 mg rifampicin (n=37). Treatment outcome was assessed by fever clearance time (the time for oral temperature to fall below 37.3 degrees C). FINDINGS: About 12,800 fever patients were screened during the 3-year study to recruit 126 patients with confirmed scrub typhus and no other infection, of whom 86 completed therapy. Eight individuals received the combined regimen that was discontinued after 1 year. The median duration of pyrexia was significantly shorter (p=0.01) in the 24 patients treated with 900 mg daily rifampicin (fever clearance time 22.5 h) and in the 26 patients who received 600 mg rifampicin (fever clearance time 27.5 h) than in the 28 patients given doxycycline monotherapy (fever clearance time 52 h). Fever resolved in a significantly higher proportion of patients within 48 h of starting rifampicin (900 mg=79% [19 of 24], 600 mg=77% [20 of 26]) than in patients treated with doxycycline (46% [13 of 28]; p=0.02). Severe gastrointestinal events warranted exclusion of two patients on doxycyline. There were two relapses after doxycycline therapy, but none after rifampicin therapy. INTERPRETATION: Rifampicin is more effective than doxycycline against scrub-typhus infections acquired in northern Thailand, where strains with reduced susceptibility to antibiotics can occur.

Transmission of Orientia tsutsugamushi, the aetiological agent for scrub typhus, to co-feeding mites.

Experiments were conducted to investigate the potential for transmission of Orientia tsutsugamushi, the aetiological agent for scrub typhus, when naturally infected mite larvae were co-feeding with uninfected larvae. Larvae from colonies of Leptotrombidium deliense and L. imphalum infected with O. tsutsugamushi were used. Transmission of O. tsutsugamushi to previously uninfected L. deliense and Blankaartia acuscutellaris co-fed with infected L. deliense was shown to occur. The overall minimum rate of acquisition was 1.6% (4/258) for L. deliense and 2.5% (3/119) for B. acuscutellaris. When individual infected L. deliense were co-fed with B. acuscutellaris acquisition of O. tsutsugamushi was not detected. However, when 4 and 8 infected larvae were co-fed with B. acuscutellaris acquisition of O. tsutsugamushi was detected. Transmission of O. tsutsugamushi was not observed when uninfected L. deliense were co-fed with infected L. imphalum. This novel transmission route may explain the occurrence of rickettsiae in genera other than Leptotrombidium spp, which are considered to be the main vectors of O. tsutsugamushi.

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.

Azithromycin activities against Orientia tsutsugamushi strains isolated in cases of scrub typhus in Northern Thailand.

Azithromycin was given to mice and humans infected with strains of Orientia tsutsugamushi from northern Thailand, where drug-resistant scrub typhus occurs. Azithromycin and doxycycline yielded comparable mouse survival rates (73 and 79%, respectively; P > 0.5). Symptoms, signs, and fever in two pregnant women abated rapidly with azithromycin. Prospective human trials are needed.

Occurrence of Orientia tsutsugamushi in chiggers (Acari: Trombiculidae) and small animals in an orchard near Bangkok, Thailand.

The ecology of Orientia tsutsugamushi (Hayashi) was studied in chiggers and small animals in an orchard near Bangkok, Thailand. Small animals were trapped monthly between July and November 1992 and examined for the presence of O. tsutsugamushi and ectoparasitic chiggers. A total of 40 Rattus rattus (L.) and 16 Tupaia glis (Diard) was trapped. O. tsutsugamushi was isolated from liver and spleen samples of 30.8% of R. rattus and 18.6% of T. glis. Antibodies to O. tsutsugamushi were detected in 95% of R rattus, and IgG antibodies persisted for up to 10 mo after removing rats from potential reinfection in the field. A total of 1,133 chiggers was identified and examined for the presence of O. tsutsugamushi using a direct fluorescent antibody test, and 2.6% Leptotrombidium deliense Walch, 5.1% Aschoshoengastia indica Hirst, 2.6% A. (Laurentella) sp. #2 and 0.9% A. (Laurentella) sp. #4 were infected. Forty-four pools of chiggers from these animals were triturated and injected into mice. Seven pools were obtained from T. glis and 1 was positive for O. tsutsugamushi, and 3 of 37 pools from R. rattus were positive. A proportion of the engorged chiggers collected was reared to the adult stage, and the progeny of these adults tested for the presence of O. tsutsugamushi. The progeny of 186 females was tested, and the progeny of 2 L. deliense was found to be naturally infected with O. tsutsugamushi.

A new ecology for scrub typhus associated with a focus of antibiotic resistance in rice farmers in Thailand.

Following the documentation of chloramphenicol-resistant and doxycycline-resistant strains of Orientia tsutsugamushi (Hyashi) in northern Thailand, we conducted ecological and epidemiological studies near the houses of patients hospitalized with antibiotic-resistant infections. New associations between chiggers, rodents, and O. tsutsugamushi in active rice agriculture areas, an ecological habitat not described previously, are reported. Rattus rattus (L.) was the most common species (representing 85.8% of the 1,433 rodents processed), followed by Rattus losea (Swinhoe) (9.4%), Bandicota indica (Bechstein) (3.6%), and Rattus argentiventer (Robinson and Kloss) (1.3%). O. tsutsugamushi was isolated from 30% of the R. rattus and R. losea, 29% of the B. indica, and 33% of the R. argentiventer collected. Mean minimum infection rates were 0.03 in Leptotrombidium chiangraiensis Tanskul & Linthicum, a new species of chigger, and 0.002 in Leptotrombidium imphalum (Vercammen-Grandjean & Langston), a chigger species not previously associated with scrub typhus transmission. Efficient vertical and horizontal transmission of O. tsutsugamushi by L. chiangraiensis and L. imphalum was demonstrated. During a 19-mo period from October 1993 to April 1995, the overall prevalence of human IgM and IgG antibody to O. tsutsugamushi was 25.5 and 47.3%, respectively. L. chiangraiensis and L. imphalum are incriminated as vectors of O. tsutsugamushi in a rice field habitat associated with a focus of antibiotic resistance.

Evaluation of an enzyme-linked immunosorbent assay in Thai scrub typhus patients.

We report the development of an improved enzyme-linked immunosorbent assay (ELISA) to detect Orientia (formerly Rickettsia) tsutsugamushi antibody in human sera. Results were compared with a standard test, the indirect immunoperoxidase assay (IIP). Control serum samples were collected from 96 American soldiers and 198 Royal Thai Army soldiers with no recent history of clinical illness. Sera were examined from 79 febrile, Thai scrub typhus patients presenting at Chiang Rai (76) and Bangkraui Nontaburi (3) Provincial hospitals (cases confirmed by elevated IIP IgG levels > or = 1:1,600, IgM levels > or = 1:400, or presence of an eschar). The mean + 2 SD, used for the upper limit of normal reactions in the IgG ELISA, was 0.10 for U.S. soldiers and 0.42 for Thai soldiers. Using the 0.10 cutoff value, 29% of the asymptomatic Thai soldiers would be designated as antibody positive. Variability of IgG ELISA values was greater in the Thai soldier group than in American soldiers, possibly reflecting previous exposure to O. tsutsugamushi. In the Thai patients, there was a significant correlation between IIP titers and single serum dilution (1:100) ELISA values (IgG, r = 0.75, n = 104; P < 0.0005; IgM, r = 0.70, n = 75; P < 0.0005) and between IIP titers and ELISA titers (IgG, r = 0.87, n = 103; P < 0.0005; IgM, r = 0.76, n = 75; P < 0.0005). The single serum dilution ELISA was as effective as the titration in determining presence of specific antibodies. The O. tsutsugamushi ELISA is a rapid and objective test amenable to accurately testing the large numbers of sera often obtained in seroepidemiologic investigations.

Scrub typhus infections poorly responsive to antibiotics in northern Thailand.

BACKGROUND: Rickettsia tsutsugamushi, the aetiological agent of scrub typhus, is common in Asia and readily infects visitors to areas where disease transmission occurs. Rapid defervescence after antibiotic treatment is so characteristic that it is used as a diagnostic test for R tsutsugamushi infection. Reports from local physicians that patients with scrub typhus in Chiangrai, northern Thailand responded badly to appropriate antibiotic therapy prompted us to do a prospective clinical evaluation and antibiotic susceptibility testing of human rickettsial isolates. METHODS: The clinical response to doxycycline treatment in patients with early, mild scrub typhus in northern Thailand was compared with the results of treatment in Mae Sod, western Thailand. Prototype and naturally occurring strains of R tsutsugamushi were tested for susceptibility to chloramphenicol and doxycycline in mice and in cell culture. FINDINGS: By the third day of treatment, fever had cleared in all seven patients from Mae Sod, but in only five of the 12 (40%) from Chiangrai (p < 0.01). Median fever clearance time in Chiangrai (80 h; range 15-190) was significantly longer than in Mae Sod (30 h; range 4-58; p < 0.005). Conjunctival suffusion resolved significantly more slowly in Chiangrai (p < 0.05). Antibiotics prevented death in mice infected by Chiangrai strains of R tsutsugamushi less often than after infection by the prototype strain (p < 0.05). Only one of three Chiangrai strains tested in cell culture was fully susceptible to doxycycline. INTERPRETATION: Chloramphenicol-resistant and doxycycline-resistant strains of R tsutsugamushi occur in Chiangrai, Thailand. This is the first evidence of naturally occurring antimicrobial resistance in the genus Rickettsia.

Pathology of Rickettsia tsutsugamushi infection in Bandicota savilei, a natural host in Thailand.

Following rodent surveys in a rice-growing area of central Thailand where we found Bandicota savilei, B. indica, and Rattus rattus infected with Rickettsia tsutsugamushi, we performed a study of pathogenesis of R. tsutsugamushi in laboratory-reared B. savilei. Eight animals were injected with saline and 19 animals were injected with 4.0 x 10(6) mouse 50% lethal dose units of a strain of R. tsutsugamushi isolated from a human in central Thailand. Animals were evaluated at intervals for IgG and IgM antibodies to R. tsutsugamushi by an indirect immunoperoxidase assay, the presence of the pathogen in liver and spleen by murine inoculation, and the pathology of representative tissues by gross and microscopic examination. The infected animals began to show internal evidence of mild illness 7-14 days after inoculation, and exhibited no changes in behavior. Total white blood cell counts decreased on day seven (including lymphocytes and polymorphonuclear leukocytes), followed by an almost equal increase on day 14. Gross pathology noted at necropsy was limited to slight liver and spleen enlargement accompanied by low numbers of abscesses and fibrinous tags present in the abdominal cavity. In addition to the gross morphologic changes, histopathologic lesions noted were all mild, consisting of vasculitis of the lung, activation of the mononuclear phagocyte system, abdominal mesothelial cell hyperplasia, and peritonitis. Rickettsiae were isolated from liver and spleen on days 0, 7, and 14, but not thereafter. Specific antibody response was first observed on day 14, peaked on day 21, and it decreased to levels observed in uninfected animals between days 120 and 180.(ABSTRACT TRUNCATED AT 250 WORDS)

Scrub typhus in suburban Bangkok: first cases.

Three patients, two of whom had eschars, were admitted with fulminant febrile disease suggestive of scrub typhus. Elevated IgG and IgM to Rickettsia tsutsugamushi were detected in all three by IFA. The cases lived in widely separated areas of western Bangkok where fruit trees are grown and claimed not to have left the areas of their homes during the estimated incubation periods. A field survey collected rodents, treeshrews, and mites fro around the homes of each case. R. tsutsugamushi was isolated from one rat and known vector species of mites were found. These are the first confirmed cases of scrub typhus transmission in Metropolitan Bangkok. It is suggested that typhus incidence around Bangkok may be substantial but has been unreported because of widespread self-medication with antibiotics and the difficulties of laboratory diagnosis.