Population pharmacokinetics of the new antimalarial agent tafenoquine in Thai soldiers.

AIMS: To describe the population pharmacokinetics of tafenoquine in healthy volunteers after receiving tafenoquine for malaria prophylaxis. METHODS: The population consisted of 135 male Thai soldiers (mean age 28.9 years; weight 60.3 kg). All soldiers were presumptively treated with artesunate for 3 days plus doxycycline for 7 days to remove any pre-existing malaria infections. After the treatment regime, 104 soldiers (drug group) received a loading dose of 400 mg tafenoquine base daily for 3 days followed by 400 mg tafenoquine monthly for 5 consecutive months. In the placebo group, 31 soldiers were infected with malaria during the study period. They were re-treated with artesunate for 3 days plus doxycycline for 7 days followed by a loading dose of 400 mg tafenoquine daily for 3 days and then 400 mg tafenoquine weekly for prophylaxis. Blood samples were randomly collected from each soldier on monthly and weekly prophylaxis. Plasma tafenoquine concentrations were measured by h.p.l.c. Population pharmacokinetic modelling was performed using NONMEM. RESULTS: A one-compartment model was found best to describe the pharmacokinetics of tafenoquine after oral administration. Age and weight influenced volume of distribution (V/F), and subjects who contracted malaria had higher clearance (CL/F), but none of these factors was considered to have sufficient impact to warrant change in dosing. The population estimates of the first-order absorption rate constant (Ka), CL/F and V/F were 0.694 h(-1), 3.20 l h(-1) and 1820 l, respectively. The intersubject variability in these parameters (coefficient of variation, CV%) was 61.2%, 25.3% and 14.8%, respectively. The absorption and elimination half-lives were 1.0 h and 16.4 days, respectively. The residual (unexplained) variability was 17.9%. CONCLUSIONS: The population pharmacokinetics of orally administered tafenoquine have been determined in Thai soldiers under field conditions. This information, together with its known potent antimalarial activity, portends well for the application of tafenoquine as a useful prophylactic drug or for short-term radical treatment of vivax malaria.

Malaria prophylaxis/radical cure: recent experiences of the Australian Defence Force.

Since the eighties, the Australian Defence Force has deployed soldiers in malaria-endemic areas: Cambodia, Somalia, Rwanda, Bougainville, and East Timor. Currently, doxycycline is used as first line prophylactic drug and mefloquine is recommended for those who cannot tolerate the antibiotic. In 1998, the Australian Defence Force participated in the evaluation of tafenoquine for prophylaxis of both falciparum and vivax malaria in Thai soldiers. At the completion of this six-month study, 29 of 205 soldiers had come down with malaria including eight with falciparum malaria, 20 with vivax malaria, and one with mixed infection. A total of 28 of the 101 soldiers in the placebo group were infected with malaria as compared with only one of the 104 soldiers in the tafenoquine group. In 1999, another study was started on the island of Bougainville to compare the effectiveness a 3-day course of tafenoquine and a 14-day course of primaquine for radical cure of vivax malaria. At the present time, 411 soldiers have completed the study including 201 in tafenoquine arm and 210 in primaquine arm. Seven soldiers in each arm developed vivax malaria after returning to Australia. These results indicate that tafenoquine is not superior to primaquine in preventing vivax malaria. However study participants preferred the shorter course using tafenoquine and operationally it was found to be more suitable than primaquine.

Evaluation of a newly developed dipstick test for the rapid diagnosis of scrub typhus in febrile patients.

Scrub typhus is a potentially fatal, febrile disease prevalent in rural Asia. The etiological agent, Orientia tsutsugamushi, is transmitted to humans by the bite of a larval trombiculid mite. No current diagnostic test is sufficiently practical for use by physicians working in rural areas. A new dipstick test using a dot blot immunoassay format has been developed for the serodiagnosis of scrub typhus. We evaluated this test on 83 patients presenting with acute fever of unknown origin at Maharaj Hospital, a tertiary care medical center in Nakhon Ratchasima, Northeast Thailand. The diagnosis of scrub typhus was confirmed in 30 of these patients (36%) by the indirect immunoperoxidase test. The sensitivity of the test was 87% and its specificity was 94%. The dot blot immunoassay dipstick is accurate, rapid, easy to use, and relatively inexpensive. It appears to be the best currently available test for diagnosing scrub typhus in rural areas where this disease predominates.

Comparative pharmacokinetics and effect kinetics of orally administered artesunate in healthy volunteers and patients with uncomplicated falciparum malaria.

The single-dose pharmacokinetics of 100 mg of orally administered artesunate (AS) were studied in 6 patient volunteers with uncomplicated falciparum malaria and in 6 healthy volunteers. Plasma concentrations of both the parent drug, AS, and its major metabolite, dihydroartemisinin (DHA), were measured simultaneously by high-performance liquid chromatography (HPLC) with electrochemical detection (ECD). The antimalarial activity of each plasma sample measured by an in vitro bioassay (BA) was used to derive activity concentrations. Artesunate was absorbed rapidly and then almost completely hydrolyzed to DHA in patients, whereas hydrolysis was incomplete in healthy volunteers. The mean +/- standard deviation (SD) maximum concentration (Cmax) of AS was 296+/-110 nmol/L, the time to peak blood level (tmax was 0.71+/-0.66 hr, the half-life (t1/2,z) was 0.41+/-0.34 hr, and the bioavailability over 12 hr (area under the curve [AUC](0-12)) was 253+/-185 nmol hr/L. Measured by HPLC, the Cmax and AUC(0-12) values of DHA in patients with malaria were significantly greater than in volunteers (1,948+/-772 and 1,192+/-315 nmol/L; 4,024+/-1,585 and 1,763+/-607 nmol hr/L, respectively; P < or = 0.05). These differences were even greater when measured by BA. The Cmax for patients with malaria was 2,894+/-2,497 and 795+/-455 nmol/L for volunteers, and AUC(0-12) was 5,970+/-3,625 and 1,307+/-391 nmol hr/L, respectively (P < or = 0.05). In contrast, DHA parameter estimates for t1/2,z and tmax were similar between patients and healthy volunteers, with values of 0.80+/-0.30 versus 0.87+/-0.06 hr and 1.50+/-0.55 versus 1.13+/-0.52 hr, respectively (P > 0.5). Both drug metabolism and tissue protein binding could contribute to the differences between the antimalarial activity of artemisinin drugs in healthy volunteers and malaria infected patients.

Measurement of tafenoquine (WR 238605) in human plasma and venous and capillary blood by high-pressure liquid chromatography.

A simple, rapid, and accurate high-pressure liquid chromatographic method with fluorescence detection is described for the measurement of tafenoquine (TQ) (also known as WR 238605) from human plasma and venous and capillary blood. Tafenoquine was measured in plasma and venous blood following protein precipitation. Chromatographic separation was achieved using a Waters S5P Spherisorb phenyl analytical cartridge (150 mm x 4.6 mm I.D., 5 microm particle size) (Waters, Milford, MA, USA) and a mobile phase of 22 mM ammonium acetate, pH 4:acetonitrile (45:55, vol/vol). The flow rate was 1.5 mL/min and the retention times were approximately 3.5 min for WR VIIIAc (internal standard) and approximately 7.8 min for TQ. The interday and intraday coefficients of variation of TQ over a concentration range of 20-1000 ng/mL in plasma were < or =8.4% and in venous blood were < or =9.6%. The mean percent difference between added concentration and obtained concentration was 7.3% in plasma and 8.5% in venous blood over the corresponding concentration range. The limit of quantitation for both fluids was 10 ng/mL. Tafenoquine concentrations were comparable between capillary and venous blood with no significant difference between measurement in both biological fluids. The clinical application of the method was demonstrated by measuring plasma and whole blood concentrations of TQ from participants in a chemosuppression trial of the drug against malaria infections in Thailand.

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

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.

Antibodies to Orientia tsutsugamushi in soldiers in northeastern Thailand.

The prevalence and incidence of antibodies to Orientia tsutsugamushi, the etiologic agent of scrub typhus, in Thai soldiers living and working near the Thai-Cambodian border in Si Sa Ket Province was investigated. The point prevalence of antibodies varied from 0 to 4.1%. The incidence of antibodies, calculated from individuals who seroconverted following a negative result in a previous bleeding 3 to 5 months earlier, was 4.21% (9/214) in January 1992, 0 in April 1992 and 3.76% (8/213) in September 1992. An annual infection rate of 2.66% was estimated.

Antibodies to Orientia tsutsugamushi in Thai soldiers.

Thai soldiers who were conscripted, Royal Thai Army forces, professional Border Patrol Police, or local militia (Thai Rangers) located in any of seven provinces of Thailand were bled in April and again, four months later, in July 1989. In 1991, soldiers from five different locations in southern Thailand were bled once, in July. Serum samples were tested by indirect fluorescent antibody assay for antibody to Orientia (formerly Rickettsia) tsutsugamushi, etiologic agent of scrub typhus, with any titer > or = 1:50 considered positive. Prior to field exercises, prevalence of antibody varied significantly between different types of units, ranging between 18.6% for Thai Rangers and 6.8% for the Royal Thai Army. The April prevalence, July prevalence, and incidence varied significantly by province in 1989, with highest incidence being 14.5% in Kanchanaburi and the lowest 0% in Utraladit. The prevalence in southern Thailand in 1991 varied between 1.6% and 6.8%. The data demonstrate that O. tsutsugamushi is widely distributed in Thailand and that military activity consisting of field exercises that simulate combat conditions significantly expose soldiers to infection.

Plasmodium falciparum and P. vivax circumsporozoite proteins in anophelines (Diptera: Culicidae) collected in eastern Thailand.

In total, 414 anophelines, consisting of Anopheles karwari (James), An. splendidus Koidzumi, An. dirus Peyton & Harrison, and An. barbirostris Van der Wulp were collected in 2 provinces in eastern Thailand and tested by enzyme-linked immunosorbent assay for the presence of circumsporozoite proteins. Plasmodium vivax CS protein was detected in 3.4% (2/54) of An. karwari and 4.8% (2/42) of An. barbirostris specimens. Both P. vivax phenotypes, Pv247 and Pv210, were found in An. karwari, whereas only Pv247 was detected in An. barbirostris. Plasmodium falciparum CS protein was detected only in 0.3% (1/276) of An. dirus. Results indicate that An. barbirostris may play a role in the transmission of P. vivax in Chanthaburi Province, Thailand.

Effectiveness of repellent formulations containing deet against mosquitoes in northeastern Thailand.

Formulations of the repellent diethyl methyl benzamide (deet) were evaluated against mosquitoes in field tests conducted in Sisaket Province, northeastern Thailand. Thirty-three percent deet in an applicator stick provided > or = 87.1% protection against primarily Culex vishnui, Cx. gelidus, and Cx. tritaeniorhynchus for up to 5 h, and 50% deet in ethanol provided > or = 93.3% protection against Anopheles sp. and culicines for up to 8 h. The U.S. Army Extended Duration Repellent Formulation ([EDRF], containing 33% deet), 50% deet in ethanol, and 75% deet in ethanol provided complete protection for up to 6 h against primarily Culex vishnui and Anopheles dirus.

Evaluation of permethrin-treated military uniforms for personal protection against malaria in northeastern Thailand.

A trial to compare the effect of military clothing treated by high-pressure spray with permethrin or placebo on the incidence of malaria in Royal Thai Army troops was conducted in northeastern Thailand. Bioassays of treated clothing using laboratory-reared Anopheles dirus females showed permethrin remained in the treated fabric for up to 90 days. Both permethrin- and placebo-treated uniform shirts provided > 84% protection from biting An. dirus in laboratory bioassays for the duration of the study. In laboratory tests, knockdown of An. dirus exposed to permethrin-treated cloth fell to < 20% after 3 hand washes, despite the presence of 28.7-59.9% of the original dose of permethrin. The use of permethrin-treated uniforms without adjunct application of topical repellents did not reduce malaria in Thai troops in an operational setting where incidence during 6 months was as high as 412 cases/1,000 in spite of chemoprophylaxis and use of untreated bednets.

Prevalence of antibodies to rickettsiae in the human population of suburban Bangkok.

Following a report of three cases of scrub typhus in suburban Bangkok, we performed a serosurvey in the patients' communities. Both IgG and IgM antibodies were measured in an indirect immunoperoxidase assay, using separate spots of antigen from Rickettsia tsutsugamushi (scrub typhus), R. typhi (murine typhus), and TT-118 spotted fever group rickettsiae. Of 215 people donating blood, antibody levels indicative of most recent exposure to R. tsutsugamushi were most prevalent (21%), followed by R. typhi (8%), and TT-118 (4%). Seroprevalence suggesting most recent exposure to R. tsutsugamushi varied by location (range 13-31%), gender (26% of females and 13% of males), age (61-80-year-old individuals were the highest, with a prevalence of 38%), and contact with orchards and orchid farms (29% of those with extensive contact, 38% of those with occasional contact, and 10% of those with no contact). These patterns indicated that exposure to R. tsutsugamushi was related to occupation and behavior, as has been observed in areas of rural transmission. Expansion of metropolitan Bangkok has created a situation in which people employed in agriculture live with people employed in the city. As a result, a serosurvey in suburban Bangkok reveals evidence of murine typhus, which is usually transmitted in urban areas, as well as scrub and tick typhus, which are usually transmitted in rural areas.

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.

Response of Anopheles dirus and Aedes albopictus to repellents in the laboratory.

Laboratory tests were conducted to study the response of Anopheles dirus and Aedes albopictus to repellent formulations containing diethyl methylbenzamide (deet) and dimethyl phthalate. Anopheles dirus was tolerant of low concentrations of deet (5-20%), and formulations containing < or = 35% deet provided protection for < or = 90 min. In contrast, Ae. albopictus was sensitive to these formulations, which provided > or = 180 min protection from bites.

Prevention of Plasmodium falciparum malaria by Fansimef and Lariam in the northeastern part of Thailand.

Two studies were conduct in Thailand in order to find appropriate falciparum malaria prophylactic drug regimens. The first study was done during June - September 1987 with 363 soldiers who received Fansimef (MSP) 1 tab/week (group 1), 337 soldiers who received MSP 1 tab/2 week (group 2) and 165 soldiers who received chloroquine 300 mg base weekly plus Fansidar 1 tab/week (group 3). At the end of the study there were 9 and 13 falciparum malaria episodes in groups 1 and 2, respectively, with incidence rates of 0.8 and 1.8 cases/100 person-months (P-M). In group 3, the corresponding values were 30 episodes and an incidence of 7.2/100 P-M. For the second study which lasted from October 1987 - January 1988 in the same area, 498 soldiers were given Fansimef 1/2 tab/week (group 4), 499 soldiers were given Lariam 1/2 tab/week (group 5) and 247 soldiers were given chloroquine plus Fansidar (group 6). Thirty malaria episodes were found in group 4, for an incidence of 2.0/100 P-M. In group 5, 23 episodes were found, for an incidence of 1.6/100 P-M. In group 6, 74 episodes occurred, ie an incidence of 12.2/100 P-M. The incidence rates of malaria among Fansimef 1 tab weekly, Fansimef half dose weekly or Lariam half dose weekly were not significantly different but were different from chloroquine plus Fansidar groups. Adverse events in each group were mild.

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.

New paper enzyme-linked immunosorbent technique compared with microimmunofluorescence for detection of human serum antibodies to Rickettsia tsutsugamushi.

A new paper enzyme-linked immunosorbent assay was developed for the screening and titration of human serum antibodies against the scrub typhus rickettsia, Rickettsia tsutsugamushi. The objective [corrected] was to provide a relatively simple method for antibody screening which required neither sophisticated laboratory equipment nor a high degree of technological skill. The technique develops an enzyme product from filter paper saturated with a 5-aminosalicylic acid substrate and enzymatically reacted with a commerically available anti-human immunoglobulin G peroxidase conjugate. The product of the enzymatic reaction can be interpreted visually. Comparison of 351 human sera tested by the immunofluorescent and paper enzyme-linked immunosorbent assays against a three-antigen pool of the Karp, Kato, and Gilliam strains of R. tsutsugamushi demonstrated an agreement of 96%. The sensitivity of the paper enzyme-linked immunosorbent assay as compared to immunofluorescence was 98.2%, and the specificity was 94.4%.