Pharmacokinetics of high-dose oseltamivir in healthy volunteers.

The effects of loading doses and probenecid coadministration on oseltamivir pharmacokinetics at four increasing dose levels in groups of eight healthy adult Thai volunteers (125 individual series) were evaluated. Doses of up to 675 mg were well-tolerated. The pharmacokinetics were dose linear. Oseltamivir phosphate (OS) was rapidly and completely absorbed and converted (median conversion level, 93%) to the active carboxylate metabolite. Median elimination half-lives (and 95% confidence intervals [CI]) were 1.0 h (0.9 to 1.1 h) for OS and 5.1 h (4.7 to 5.7 h) for oseltamivir carboxylate (OC). One subject repeatedly showed markedly reduced OS-to-OC conversion, indicating constitutionally impaired carboxylesterase activity. The coadministration of probenecid resulted in a mean contraction in the apparent volume of distribution of OC of 40% (95% CI, 37 to 44%) and a reduction in the renal elimination of OC of 61% (95% CI, 58 to 62%), thereby increasing the median area under the concentration-time curve (AUC) for OC by 154% (range, 71 to 278%). The AUC increase for OC in saliva was approximately three times less than the AUC increase for OC in plasma. A loading dose 1.25 times the maintenance dose should be given for severe influenza pneumonia. Probenecid coadministration may allow considerable dose saving for oseltamivir, but more information on OC penetration into respiratory secretions is needed to devise appropriate dose regimens.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine.

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.

Lymphocyte responsiveness to a candidate malaria sporozoite vaccine (R32tet32) of individuals with naturally acquired Plasmodium falciparum malaria.

Lymphocyte proliferative responses to the candidate malaria sporozoite vaccine antigen R32tet32 were evaluated in 29 patients with acute Plasmodium falciparum malaria, 20 convalescent patients, 11 nonimmune individuals, and 22 healthy residents of two endemic malarious areas in Thailand. The results indicate that 14 of 20 (70%) convalescent patients and 14 of 22 (64%) residents of endemic areas responded to the R32tet32 antigen. However, only 8 of 29 (28%) patients with acute P. falciparum malaria responded. When 4 of the convalescent patients who remained in a malaria-free area were restudied 5-10 months after the acute infection, they were either not responsive or their responses had greatly diminished. These findings show that sensitization to R32tet32 occurs following a natural P. falciparum infection, but the cellular immune response to sporozoite antigens may be short-lived and may be suppressed during acute P. falciparum malaria.

Reduced hepatic blood flow and intestinal malabsorption in severe falciparum malaria.

We have studied intestinal function and liver blood flow in Thai adults with complicated and uncomplicated falciparum malaria. The absorption of 3 orally administered test sugars, D-xylose, 3-O-methyl-D-glucose, and L-rhamnose, was greatly reduced in complicated malaria, while the lactulose/rhamnose absorption ratio was significantly increased. Hepatic blood flow was concomitantly reduced in severe malaria. These deviations reverted to normal in convalescence. Neither sugar absorption nor liver blood flow was reduced in uncomplicated falciparum malaria.

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice.

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora.

Convulsions in childhood malaria.

A retrospective survey was conducted of all 2911 children admitted with malaria to 4 provincial hospitals in eastern Thailand between 1977 and 1987. 96 (3.3%) had cerebral malaria of whom 21 (22%) died, 225 (7.7%) had convulsions but were not comatose (4 died), and 2590 were conscious and had no fits (5 died). Thus the relative risk of a fatal outcome associated with convulsions, in the absence of cerebral malaria, was 9.2 (95% confidence interval [CI] = 2.5-34.1), P = 0.004. Overall, Plasmodium falciparum caused 81% of infections, P. vivax 16%, and 3% were mixed. Convulsions without cerebral malaria were more common in children under 3 years old (16%) compared with older children (3%): relative risk 5.6 (95% CI = 4.2-7.5), and were significantly associated with falciparum malaria (8.3%) compared with vivax malaria (4.7%): relative risk 1.7 (95% CI = 1.1-2.7). Convulsions are an important complication of malaria in young children, and are associated specifically with P. falciparum infection, even in otherwise uncomplicated malaria.

Amoxycillin-clavulanic acid treatment of melioidosis.

Melioidosis is a serious infection with high acute mortality, and a high rate of relapse despite protracted antimicrobial treatment. The current recommended conventional oral treatment regimen is a 4-drug combination of high-dose chloramphenicol, doxycycline and trimethoprim-sulphamethoxazole given for between 6 weeks and 6 months. We have evaluated prospectively the use of amoxycillin-clavulanic acid, to which Pseudomonas pseudomallei is consistently sensitive in vitro, for the oral maintenance treatment of melioidosis. Amoxycillin-clavulanic acid was used either as sole treatment of localized disease, or as maintenance therapy following either parenteral ceftazidime or the conventional 4-drug regime; 20 patients with localized infections and 26 with septicaemic melioidosis received a median of 7.5 (2-12) weeks treatment. After a mean follow-up period of 6 months (range 1-19), 31 patients (67%) remain free of disease. The drug was well tolerated. Three patients had fatal relapses, one other died suddenly at home, and another died from underlying promyelocytic leukaemia. The remaining 10 relapses were treated successfully. Resistance developed in one case. Amoxycillin-clavulanic acid is a safe alternative to the conventional 4-drug antimicrobial combination for the oral treatment of melioidosis. It may be of particular value in children, pregnant women, and in infections with Ps. pseudomallei resistant to the potentially toxic conventional regimen, but the optimum dose and duration of therapy need to be established.

The use of bone marrow culture for the diagnosis of melioidosis.

We have evaluated prospectively the contribution of bone marrow culture to the diagnosis of melioidosis. Bone marrow (BMC) and blood cultures (BC) were collected concurrently from 105 patients with suspected acute, severe melioidosis. 67 patients were subsequently proved to have the disease whilst other significant organisms were isolated from these specimens in 5 cases. Overall, 67.2% of BC and 64.2% of BMC from melioidosis patients grew Pseudomonas pseudomallei. Time to positivity did not differ significantly in paired BC and BMC specimens. These results do not support the routine use of BMC in the diagnosis of acute, severe melioidosis. In one patient with pulmonary melioidosis, however, blood cultures were repeatedly negative, whilst bone marrow grew P. pseudomallei, and this preceded the development of a distant focus of infection. This suggests that culture of bone-marrow may be of value in certain blood culture-negative patients with melioidosis.

ParaSight-F test for the detection of treatment failure in multidrug resistant Plasmodium falciparum malaria.

The ParaSight-F test for the detection of Plasmodium falciparum was evaluated for its accuracy and usefulness in predicting treatment outcome in 75 patients (70 males, 5 females) with acute uncomplicated malaria who attended a malaria clinic in Mae Sot, Tak province, on the Thai-Myanmar border. All patients were admitted to the clinic for 28 d to exclude reinfection. The test was performed using blood samples collected into ethylenediaminetetraacetic acid from the patients on admission, and on days 1, 2, 7, and 14. The presence of microscopically detectable parasitaemia was used as the reference for sensitivity and specificity of the test. The reappearance of parasites on day 28 was used to determine the accuracy of predicting the outcome of artemether treatment on day 14. The sensitivity of the ParaSight-F test on admission, and on days 1, 2, 7, and 14, was 98.7%, 96.7%, 100%, 100% and 100%, respectively, with corresponding specificities of 50%, 24.2%, 47.1% and 72.9%. The sensitivity for predicting recrudescence by using the test on day 14 was 100%, with 97.7% specificity, and the sensitivity of predicting a sensitive response on day 14 was 97.7%, with 100% specificity. The test seems to permit more precise detection of treatment failure under 'field' conditions if used on day 14 after the start of treatment.

Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais.

Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells (PRBC) [Mol Biochem Parasitol, 76, (1996) 1], and may be involved in the pathogenic process of cerebral malaria (CM) which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1/CD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients (43 cerebral, 89 severe and 78 uncomplicated) were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine (V)-563 asparagine (N); 125V-563 serine (S); 125 leucine (L)-563N; and 125L-563S. We found that the frequency of the 125 V/V 563 N/N genotype was significantly high in CM patients as compared with severe cases without CM (P<0.01, OR=2.92), suggesting that this genotype is one of the risk factors for CM.

Axenic cultivation of Entamoeba histolytica from liver abscess and its zymodeme.

A local strain of Entamoeba histolytica, the HTH-56: MUTM from a human liver abscess was successfully axenized. The culture was initially established monoxenically in Diamond's TYI-S-33 medium in the presence of Crithidia luciliae and maintained at 34 +/- 0.5 degrees C. After 5 passages it was adapted to axenic cultivation by addition of 0.02% Bacto agar in Diamond's TYI-S-33 medium in place of Crithidia. Subcultures or replacement with fresh complete media were done twice or thrice for 7 days, after which the agar was omitted and a stable culture was obtained. Isoenzyme analysis showed that this strain of E. histolytica belonged to the zymodeme II pattern, which is one out of 10 pathogenic zymodemes of E. histolytica most commonly found among the virulent strains.

Activity of artemether-azithromycin versus artemether-doxycycline in the treatment of multiple drug resistant falciparum malaria.

The efficacy of the combination of artemether with doxycycline or azithromycin was evaluated in 60 patients with acute uncomplicated falciparum malaria who attended malaria clinic in Mae Sot, Tak Province (Thai-Myanmar border). Patients (30 each) were randomized to receive (a) 300 mg artemether together with 100 mg doxycycline as initial doses, followed by 100 mg artemether plus 100 mg doxycycline at 12 hours later, then 100 mg doxycycline every 12 hours for another 4 days, or (b) 300 mg artemether together with 500 mg azithromycin, followed by 250 mg azithromycin at 24 and 48 hours. The follow-up period was 28 days. Patients in either group had a rapid initial response to treatment with comparable PCT and FCT. The cure rate of artemether-azithromycin regimen was significantly lower than that of artemether-doxycycline regimen (14.8 vs 53.3%). Low cure rate from artemether-azithromycin combination in this study was likely to be due to inadequate azithromycin dosage. However, with the low incidence of gastrointestinal adverse effects, the once daily dose of azithromycin could still be increased in order to enhance its clinical efficacy. The simplicity of drug administration and lesser incidence of adverse effects make azithromycin a more proper partner of artemether than doxycycline. Further dose-finding and pharmacokinetic study with the artemether-azithromycin combination is encouraging.

Blood culture techniques for the diagnosis of melioidosis.

The effects of variations in laboratory technique on the speed and sensitivity of isolation of Pseudomonas pseudomallei from blood were evaluated prospectively. Pseudomonas pseudomallei was isolated from 154 of 546 cultures from 325 patients with suspected or confirmed melioidosis. Subcultures after 12 to 24 and 36 to 48 hours of incubation were positive in 52.3% and 80.8% respectively. The yields from 20 ml (blood to broth ratio 1:4) and 50 ml (blood to broth ratio 1:10) brain heart infusion broth bottles were equivalent in patients not receiving treatment for melioidosis. During therapy, the 50 ml bottles grew Pseudomonas pseudomallei significantly faster than the 20 ml bottles (p less than 0.01), and gave a higher overall yield for cultures processed in antimicrobial removal devices (p less than 0.05). These devices themselves increased the speed of isolation of the organism from treated patients (p less than 0.01). In most cases, all bottles collected from a patient before treatment were positive, and a single 20 ml bottle had an estimated relative sensitivity of 85.7% (95% confidence interval 77.1-94.3%). Early subculture should be employed routinely for the laboratory diagnosis of septicaemic melioidosis. However, blood culture techniques do not need to be sophisticated. Culture of 5 ml blood in 20 ml broth is a simple and sensitive procedure suitable for regions where melioidosis is currently under-diagnosed.

Halving of mortality of severe melioidosis by ceftazidime.

An open randomised trial was conducted to compare ceftazidime (120 mg/kg/day) with "conventional therapy" (chloramphenicol 100 mg/kg/day, doxycycline 4 mg/kg/day, trimethoprim 10 mg/kg/day, and sulphamethoxazole 50 mg/kg/day) in the treatment of severe melioidosis. A paired restricted sequential trial designed to detect a reduction in mortality from 80 to 40% in culture-positive patients surviving greater than 48 hours was stopped after 22 months. Of the 161 patients entered into the study, 65 had bacteriologically confirmed melioidosis and 54 of these were septicaemic. Ceftazidime treatment was associated with a 50% (95% CI 19-81%) lower overall mortality than conventional treatment (74% vs 37%; p = 0.009) and should now become the treatment of choice for severe melioidosis.

Pharmacokinetics of chloroquine in Thais: plasma and red-cell concentrations following an intravenous infusion to healthy subjects and patients with Plasmodium vivax malaria.

1. Chloroquine diphosphate (15 mg base kg-1) was given by constant rate intravenous infusion to two groups of Thai subjects. Eleven were patients with malaria (10 with Plasmodium vivax and one case with Plasmodium malariae) and 10 were healthy normal volunteers. 2. Plasma and packed red-cell concentrations of chloroquine, electrocardiographic intervals, arterial blood pressure and pulse were measured at frequent intervals. 3. Peak plasma concentrations at the end of the infusion ranged from 979 to 2,900 ng ml-1 in the malaria patients. In the group of healthy subjects the range was 550-2,200 ng ml-1. Values for terminal elimination rate constant, (lambda z) plasma clearance (CL), initial volume of distribution (V1) and volume of distribution at steady state (Vss) were calculated. For the healthy subjects, mean estimates of these parameters were lambda z = 0.062 +/- 0.030 day-1, CL = 597 +/- 238 ml min-1, V1 = 0.66 +/- 0.71 l kg-1 and Vss = 132 +/- 50 l.kg-1 For the group of malaria patients, the corresponding values were lambda z = 0.055 +/- 0.032 day-1, CL = 535 +/- 246 ml min-1, V1 = 0.74 +/- 0.75 l kg-1 and Vss = 136 +/- 64 l kg-1 There was no statistically significant difference in the estimates for any parameter between groups (P less than or equal to 0.05). 4. Chloroquine concentrations in packed red blood cells consistently exceeded those in plasma and showed no consistent change with time throughout the period of study in either group. The median value for the red cell to plasma ratio was between 3 and 4 in each group.(ABSTRACT TRUNCATED AT 250 WORDS)

Single dose pharmacokinetics of proguanil and its metabolites in healthy subjects.

1. Plasma and whole blood concentrations of proguanil and its two major metabolites cycloguanil (CG) and 4-chlorophenylbiguanide (CPB) were measured by a sensitive h.p.l.c. technique in nine healthy adult male volunteers after a single oral dose of proguanil 200 mg. 2. Proguanil was absorbed with a median time to peak plasma concentration of 3 h (range 2-4 h). 3. Peak plasma concentrations of proguanil ranged between 150 and 220 (median 170) ng ml-1 compared with 12 to 69 (median 41) ng ml-1 for the active antimalarial metabolite CG, and 3 to 16 (median 11) ng ml-1 for CPB. Peak (mean +/- s.d.) plasma CG concentrations occurred 5.3 +/- 0.9 h and peak CPB concentrations occurred 6.3 +/- 1.4 h after oral administration of proguanil. 4. Whole blood concentrations of proguanil were approximately five times higher, and whole blood CPB concentrations were four times higher than corresponding plasma values, whereas plasma and whole blood concentrations of CG were similar. 5. A triexponential function was fitted to these data; mean (+/- s.d.) values for the AUC were 3046 +/- 313 ng ml-1 h for proguanil, 679 +/- 372 ng ml-1 h for CG and 257 +/- 155 ng ml-1 h for CPB. 6. Plasma and whole blood concentrations of proguanil and its metabolites declined in parallel with terminal elimination half-lives estimated as 16.1 +/- 2.9 h and 15.7 +/- 2.4 h, respectively. Mean residence times in plasma and whole blood were estimated as 21.2 +/- 4.9 and 19.3 +/- 2.4 h.

Intramuscular loading dose of quinine for falciparum malaria: pharmacokinetics and toxicity.

In a study of intramuscular injection of quinine eight adults with moderately severe falciparum malaria resistant to chloroquine were treated with quinine dihydrochloride, being given a loading dose of 20 mg salt (16.7 mg base)/kg followed by three or four eight hourly maintenance doses of 10 mg salt (8.3 mg base)/kg injected into the anterior thigh. All patients responded to treatment. Fever and parasite clearance times (mean (SD) 60 (23) h and 53 (22) h respectively) were comparable with those obtained with intravenous quinine. The mean peak plasma quinine concentration of 11.0 mg/l (34.4 mu mol/l) [corrected] was reached a median of five hours after administration of the loading dose. In all patients plasma quinine concentrations exceeded the high minimum inhibitory concentration for Plasmodium falciparum malaria prevalent in Thailand within four hours of the start of treatment but did not cause toxicity other than mild cinchonism. When intravenous infusion is not possible an intramuscular quinine loading dose is an effective means of starting treatment in patients with moderately severe falciparum malaria who cannot swallow tablets.

Splenic Fc receptor function in host defense and anemia in acute Plasmodium falciparum malaria.

To determine splenic Fc receptor function in patients with acute Plasmodium falciparum malaria, the clearance of IgG-coated autologous 51Cr-labeled erythrocytes in 20 patients and 10 normal controls was studied. Clearance half-times were directly correlated with both the absolute parasite count (r = .635, P less than .005) and hematocrit (r = .791, P less than .001). Clearance half-times in patients varied from 1.0 to 96.3 h (median, 14.8 h) while those of controls ranged from 8.0 to 80.3 h (median, 23.1 h) (P = .10). Nine of the 20 patients had clearance half-times shorter than the lower 95% confidence limit of controls (less than 12.4 h). The clearance of IgG-coated erythrocytes was accelerated after parasites were eliminated from the circulation (P less than .05) and returned toward normal 6-8 weeks after the acute infection. Although circulating immune complexes were detectable, there was no correlation between immune complex levels and clearance half-times (P greater than .05). The failure to increase Fc receptor-mediated red cell clearance in patients with high parasitemias suggests inadequate splenic phagocytic activity in the face of considerable antigenic challenge. These findings indicate that splenic Fc receptor function may be important both in the control of infection and the development of anemia in P. falciparum malaria.

Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand.

In a prospective study of all patients with Pseudomonas pseudomallei infections admitted to a large provincial hospital in northeastern Thailand, 63 cases of septicemic melioidosis and 206 patients with other community-acquired septicemias were documented during a 1-y period. Apart from P. pseudomallei, the spectrum of bacteria isolated from blood cultures and the overall mortality (32%) were similar to those previously reported elsewhere. Death from septicemia was associated with failure to develop a leukocytosis or pyrexia over 38 degrees C, azotemia, hypoglycemia, and jaundice. Septicemic melioidosis presented mainly in the rainy season, occurred predominantly in rice farmers or their families, and was significantly associated with preexisting diabetes mellitus or renal failure (P = .03). Blood-borne pneumonia and visceral abscesses were common and the mortality was high (68%; P less than .001). The response to appropriate treatment was slow (median fever clearance time 5.5 d) and the median duration of hospital stay was 4 w. Septicemic melioidosis is a major cause of morbidity and mortality in northeast Thailand.

Antibody-dependent red cell removal during P. falciparum malaria: the clearance of red cells sensitized with an IgG anti-D.

The clearance of autologous red cells sensitized with an IgG anti-D has been studied in patients during and after an attack of P. falciparum malaria, and in 11 uninfected control subjects. In most patients with P. falciparum malaria there was evidence of increased clearance of sensitized red cells, compared to controls. Clearance half-times of IgG sensitized red cells were significantly decreased (P less than 0.01) in 16 patients with acute falciparum malaria (median 21 min, range 11-53 min) compared to 11 control subjects (median 37 min, range 20-60 min). This difference was independent of the degree of IgG sensitization of red cells. In patients with acute falciparum malaria, clearance half-times were positively correlated with venous haematocrit (P less than 0.05). In 11 patients studied between 1 and 9 weeks after the attack, clearance half-times (median 17 min, range 11-56 min) were also significantly decreased compared to controls (P less than 0.01). In the majority of acute and convalescent patients, there was further evidence for early destruction of an additional substantial proportion of sensitized red cells. Our data indicate that in most patients with uncomplicated P. falciparum malaria, IgG sensitized red cells are rapidly removed from the peripheral circulation by the spleen, and that rapid clearance persists during recovery.