Malaria in Brazilian military personnel deployed to Angola.

BACKGROUND: Malaria represents one of the most important infectious disease threats to deployed military forces; most personnel from developed countries are nonimmune personnel and are at high risk of infection and clinical malaria. This is especially true for forces deployed to highly-endemic areas in Africa and Southeast Asia where drug-resistant malaria is common. METHODS: We conducted an outbreak investigation of malaria cases in Angola where a total of 439 nonimmune Brazilian troops were deployed for a 6-month period in 1995-1996. A post-travel medical evaluation was also performed on 338 (77%) of the 439 soldiers upon return to Brazil. Questionnaire, medical record, thick/thin smear, and serum anti-Plasmodium falciparum antibody titer (by IFA) data were obtained. Peak serum mefloquine (M) and methylmefloquine (MM) metabolite levels were measured in a subsample of 66 soldiers (42 cases, 24 nonmalaria controls) who were taking weekly mefloquine prophylaxis (250 mg). RESULTS: Seventy-eight cases of malaria occurred among the 439 personnel initially interviewed in Angola (attack rate = 18%). Four soldiers were hospitalized, and 3 subsequently died of cerebral malaria. Upon return to Brazil, 63 (19%) of 338 soldiers evaluated were documented to have had clinical symptoms and a diagnosis of malaria while in Angola. In addition, 37 (11%) asymptomatically infected individuals were detected upon return (< 1% parasitemia). Elevated, post-travel anti-P. falciparum IFA titers (> or = 1:64) were seen in 101 (35%) of 292 soldiers tested, and was associated with a prior history of malaria in-country (OR = 3.67, 95% CI 1.98-6.82, p <.001). Noncompliance with weekly mefloquine prophylaxis (250 mg) was associated with a malaria diagnosis in Angola (OR = 3.75, 95% CI 0.97-17.41, p =.03) but not with recent P. falciparum infection (by IFA titer). Mean peak levels (and ratios) of serum M and MM were also found to be lower in those who gave a history of malaria while in Angola. CONCLUSIONS: Malaria was a significant cause of morbidity among Brazilian Army military personnel deployed to Angola. Mefloquine prophylaxis appeared to protect soldiers from clinical, but not subclinical, P. falciparum infections. Mefloquine noncompliance and an erratic chemoprophylaxis prevention policy contributed to this large outbreak in nonimmune personnel. This report highlights the pressing need for development of newer, more efficacious and practical, prophylactic drug regimens that will reduce the malaria threat to military forces and travelers.

Monitoring for mefloquine-resistant Plasmodium falciparum in Africa: implications for travelers' health.

The effectiveness of mefloquine to prevent malaria caused by Plasmodium falciparum is influenced by the sensitivity of the malaria parasites to this drug. Concern has been raised that resistance to mefloquine may develop in sub-Saharan Africa as has been observed in Southeast Asia. Case reports, along with blood smears to confirm the diagnosis and blood samples to determine the mefloquine concentration, were provided on any Peace Corps volunteer serving in sub-Saharan Africa who was diagnosed with malaria. We defined prophylaxis failures probably due to mefloquine resistance as patients with P. falciparum malaria confirmed at the Centers for Disease Control and Prevention, reported compliance with prophylaxis, no ingestion of mefloquine between date of illness onset and date of blood drawing, and a mefloquine level > or = 620 ng/ml in blood drawn within five days of onset of illness. Between January 1, 1991 and September 6, 1996, 44 (31%) of 140 volunteers with confirmed P. falciparum had blood drawn within five days of onset of illness. Twenty-nine (66%) had not fully complied with prophylaxis. Five of 15 prophylaxis failures in four countries had mefloquine levels > or = 620 ng/ml. Failure of mefloquine prophylaxis is primarily due to noncompliance. Evidence of probable resistance to mefloquine among strains of P. falciparum was found in five Peace Corps volunteers in sub-Saharan Africa. Clusters of well-documented prophylaxis failures need to be followed-up by therapeutic in vivo studies to document parasite resistance to mefloquine. Reduced sensitivity to mefloquine does not (yet) appear to be a significant problem in sub-Saharan Africa.

Comparison of whole blood and serum levels of mefloquine and its carboxylic acid metabolite.

Because of the widespread presence of chloroquine-resistant Plasmodium falciparum malaria, mefloquine is now the recommended drug of choice for long-term malaria prophylaxis in these areas. Although several studies have compared plasma and whole blood concentrations of either mefloquine or its carboxylic acid metabolite, we report the first comparison of serum and whole blood levels in 86 Dutch marines taking 250 mg of mefloquine weekly for 18 weeks while deployed in western Cambodia. All samples were taken during steady-state and at 42-48 hr after the most recent dose. The concentration of mefloquine in serum (mean = 979 ng/ml) was significantly greater than in whole blood (mean = 788 ng/ml) (P < 0.00001, by paired t-test) with an overall mean ratio of 1.28. The concentration of the metabolite in serum (mean = 3,039 ng/ml) was also significantly greater than in whole blood (mean = 1,390 ng/ml) (P < 0.00001, by paired t-test) with an overall mean ratio of 2.25. These findings are similar to previous reports of plasma-to-whole blood levels. Furthermore, we report that the within-individual ratios of the metabolite concentration to the mefloquine concentration were also found to be significantly different in serum (3.79; P < 0.00001, by paired t-test) and in whole blood (2.02; P < 0.00001, by paired t-test). Appropriate attention must be given to these differences when comparing serum and whole blood concentrations of either mefloquine or its metabolite to avoid misinterpretation of their respective levels. Also, the determination of the relative mefloquine ratios in various blood fluids, as well as the documentation of the metabolite levels and their ratios, is critical to the appropriate interpretation of both chemoprophylaxis and chemotherapy, especially in the presence of resistant strains.

Field detection of sulfonamides in urine: the development of a new and sensitive test.

A new, field-adapted, colorimetric method for detecting sulfonamide drugs in urine is described. The method uses the color reagent, p-dimethylaminocinnamaldehyde, and has a detection limit of about 1 microgram/ml. Analysis of 35 samples collected in the field, comparing results obtained with the colorimetric field test with those obtained using high-performance liquid chromatography, indicated a calculated sensitivity value of 94% and a specificity value of 94% for the test to detect the presence of sulfonamides. The field test can be modified to allow quantitation of sulfonamides in urine in field situations, using a hand-held, portable photometer for measuring the absorbance of test solutions. For this test, calculated coefficients of variation for day to day reproducibility were < or = 5% at sulfonamide concentrations > or = 3 micrograms/ml. This new test for detecting the presence of sulfonamides in urine is more sensitive and reliable than the presently used Bratton-Marshall test.

A correlation of the sequestration rate of Plasmodium coatneyi-infected erythrocytes in cerebral and subcutaneous tissues of a rhesus monkey.

Parasitized red blood cells (PRBCs) were sequestered in microvessels of cerebral and subcutaneous tissues of a rhesus monkey infected with Plasmodium coatneyi. A similar sequestration rate (approximately 80%) was observed in both cerebral and subcutaneous microvessels. Electron microscopy showed knobs of the sequestrated PRBCs cytoadhered to endothelial cells. These results are consistent with the finding of PRBC sequestration in subcutaneous tissues in a comatose patient with cerebral malaria. Biopsy specimens of subcutaneous tissue may be useful as indicators of PRBC sequestration in the brain of cerebral malaria patients.

Long-term malaria chemoprophylaxis with mefloquine in Dutch marines in Cambodia.

Three Dutch marine battalions (n=2289) serving in Western Cambodia during 1992-1993 used mefloquine as weekly malaria chemoprophylaxis. One battalion started with a loading dose. Full compliance with prophylaxis was reported by 86.3%, and possible mefloquine-related adverse events were reported by 30.2%. Sixty-four periods of malaria were diagnosed in 59 marines. During deployment, 31 Plasmodium falciparum and no Plasmodium vivax infections occurred. After return, there were 11 cases of falciparum malaria and 22 of vivax malaria, 16-72 days and 30-540 days, respectively, after stopping prophylaxis. Mefloquine-resistant parasites were isolated from 4 Dutch and 4 Khmer patients. Long-term mefloquine prophylaxis was well tolerated but not totally effective.

Determination of sulfadoxine concentrations in whole blood using C18 solid-phase extraction, sodium dodecyl sulfate and dimethylaminocinnamaldehyde.

A simple method is described for the extraction and subsequent analysis of sulfadoxine in human whole blood using a solid-phase extraction technique and colorimetric reaction. This procedure utilizes the micellar properties of sodium dodecyl sulfate to: (1) extract sulfadoxine from a C18 solid-phase sample-preparation column; (2) enhance the colorimetric reaction produced by the addition of p-dimethylaminocinnamaldehyde (DMAC); and (3) provide stability to the coloured product generated by the reaction of sulfadoxine with DMAC. The intense, violet-red colour reaction can be conveniently used for qualitative and semiquantitative visual interpretations of sulfadoxine levels. Under the assay conditions, drug concentrations in the blood of subjects receiving sulfadoxine were determined from absorbance measurements. These results correlated well with the sulfadoxine levels determined from high-performance liquid chromatographic analysis. Important advantages of the procedure include the ability to evaluate small samples of whole blood (100 microliters), the minimal use of organic solvents, no sophisticated instrumentation, and formation of a stable, coloured reaction product. The method proved to be a suitable field assay for determining whole-blood levels of sulfonamides in the concentration range from 5 to 100 micrograms ml-1.

Packed-column supercritical fluid chromatography of artemisinin (qinghaosu) with electron-capture detection.

In this preliminary report, a supercritical fluid chromatographic method is described for the determination of artemisinin in whole blood. The chromatography is carried out on a 20 cm x 1 mm I.D. Deltabond cyano supercritical fluid chromatographic column with detection of the artemisinin via an electron-capture detector. The sample work-up uses a liquid-liquid extraction with hexane, giving a recovery of 82%. The current limit of detection using 1 ml of blood is 20 ng/ml. We speculate that the endoperoxide moiety accounts for the response to the electron-capture detector and thus provides a new approach by which this class of compounds may be analyzed.

Chemiluminescent detection of artemisinin. Novel endoperoxide analysis using luminol without hydrogen peroxide.

A novel method for artemisinin quantitation employing high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection in the absence of hydrogen peroxide (H2O2), is reported. After elution from the HPLC column, artemisinin is combined with an alkaline solution of hematin and luminol. The resulting CL signal is detected by use of a spectrofluorometer with the excitation lamp disabled, and is proportional to artemisinin concentration. The CL method was optimized and applied to the analysis of artemisinin in spiked human serum. CL in the absence of H2O2 or other known oxidizing species is remarkable since such oxidizers are usually required to produce CL from luminol under alkaline conditions. Artemisinin, a naturally occurring sesquiterpene, is one of several natural products that contain an endoperoxide functional group. Since H2O2 is not needed in the analysis, the endoperoxide moiety on artemisinin is implicated as a contributing source of superoxide radicals required for the light-producing reaction with luminol.

Levels of non-ortho-substituted (coplanar), mono- and di-ortho-substituted polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans in human serum and adipose tissue.

We have measured non-ortho-substituted (coplanar) polychlorinated biphenyl (PCB) levels as well as polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels in human adipose tissue and serum collected in Atlanta, Georgia. The results show that the concentrations of the coplanar PCBs can be more than an order of magnitude higher than the concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Our measurements in pooled serum collected in 1982, 1988, and 1989 show a decrease in coplanar PCB levels from 1982 to 1989. We found that the pattern of relative amounts of coplanar PCBs in adipose tissue varied greatly from person to person unlike the PCDD and PCDF patterns, which were more nearly the same. Age was significantly correlated with the concentrations of 2,3,7,8-TCDD,3,3'4,4'-PCB, 3,3',4,4',5-PCB, and 3,3'4,4',5,5'-PCB in adipose tissue. We also measured levels of the mono- and di-ortho chlorine-substituted PCBs in human serum. The levels for some of these PCB congeners were three orders of magnitude higher than the coplanar PCBs, PCDDs, and PCDFs. We used the international toxicity equivalency factors (TEFs) for PCDDs and PCDFs and the TEFs proposed by Safe for PCBs to calculate the 2,3,7,8-TCDD equivalents. Four PCBs (3,3',4,4',5-; 2,3',4,4',5-;2,3,3',4,4'-;2,3,3',4,4',5-) make a larger contribution than 2,3,7,8-TCDD, while four other PCBs (3,3',4,4'5,5'-; 2,2',3,4,4',5'-;2,2',4,4',5,5'-;2,2',3,4,4',5,5'-) make nearly the same contribution as 2,3,7,8-TCDD. The mono-ortho-chlorine-substituted 2,3',4,4',5-PCB, however, is the major contributor to the total 2,3,7,8-TCDD equivalents in general population samples from the United States, Sweden, and Japan.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas chromatographic and spectral properties of pentafluorobenzyl derivatives of 2,4-dichlorophenoxyacetic acid and phenolic pesticides and metabolites.

Eleven phenols and 2,4-dichlorophenoxyacetic acid, compounds that may be found in body fluids of humans exposed to pesticides, are derivatized with pentafluorobenzyl bromide and characterized by gas chromatography with electron capture detection. These derivatives are further characterized by positive and negative chemical ionization mass spectrometry, nuclear magnetic resonance spectroscopy, and gas chromatography-Fourier transform infrared spectroscopy. Negative chemical ionization mass spectra of all derivatives have an anionic base peak derived from the parent analyte. In the positive mode the nonchlorinated derivatives have base peaks indicative of the analyte, while chlorinated derivatives are cleaved to give the pentafluorobenzyl cation as base peak. The possibility is explored that ortho-substituted phenols might be formed as byproducts in these derivatizations.

Quantification of selected herbicides and chlorinated phenols in urine by using gas chromatography/mass spectrometry/mass spectrometry.

We have developed a method for determining selected chlorinated phenols and phenoxy herbicides in urine. The process of preparing the samples includes acid hydrolysis, extraction with benzene, derivatization with diazoethane, and column chromatography cleanup. We quantify the more volatile compounds by using capillary column gas chromatography/positive chemical ionization/mass spectrometry/mass spectrometry. Less volatile compounds are quantified by using electron capture negative chemical ionization in a single stage mass spectrometry mode. Quality control samples are included in each analytical run, and the results demonstrate that the analytical system is in control. Positive values for the target analytes are determined on the basis of appropriate relative retention time, a signal-to-noise ratio greater than 3:1, and a calculated concentration greater than 1 ppb. We determine the chlorine isotope ratios for each compound to assess the presence or absence of interferences. This analytical method has been applied in a case-control study of 199 individuals to examine exposure to the 12 target analytes.

The pharmacokinetics of pentazocine and tripelennamine.

The pharmacokinetics of single and combined doses of pentazocine HCl (40 and 80 mg) and tripelennamine HCl (50 and 100 mg) were studied in six healthy drug abusers. After intramuscular administration of 40 or 80 mg pentazocine alone, mean peak plasma concentrations at 15 minutes were 102 and 227 ng/ml, respectively, and mean plasma t1/2 values were 4.6 and 5.3 hours, respectively. After intramuscular administration of 50 or 100 mg tripelennamine, mean plasma concentrations at 30 minutes were 105 and 194 ng/ml, respectively, and mean plasma t1/2 values were 2.9 and 4.4 hours, respectively. After concurrent administration of pentazocine with tripelennamine, plasma pentazocine and tripelennamine concentrations at all time points were not significantly different from those when pentazocine or tripelennamine was administered alone. Coadministration of pentazocine and tripelennamine had no effect on the distribution, elimination, and clearance of either pentazocine or tripelennamine. In conclusion, there did not appear to be a clinically significant metabolic interaction between pentazocine and tripelennamine.

Nature of nicotine binding to rat brain P2 fraction.

(-)-Nicotine may bind to as many as 5 sites in the rat brain P2 preparation: A very high affinity site (KD approximately 2.2 X 10(-11) M); a positive cooperativity site; a high affinity site (KD approximately 5.2 X 10(-9) M); a low affinity site (KD approximately 4.5 X 10(-5) M) and a very low affinity site. The curvilinear nature of both Scatchard plots and kinetic curves indicates the presence of multiple binding sites. Evidence for a positive cooperativity site includes: (1) The configuration of Scatchard plots (at low concentrations) of saturation as well as inhibition curves for (-)- and (+)-nicotine. (2) The Hill number of 1.37 for the binding of low concentrations of (+/-)-[3H]nicotine. (3) Selectivity among cholinergic drugs for producing positive cooperativity. (4) Markedly different specificities of drugs for the positive cooperativity site. Thus while only (+)- and (-)-nicotine interacted with the very high affinity site, acetylcholine, atropine, mecamylamine, lobeline, carbachol, (+)-nicotine and (-)-nicotine enhanced the binding of (+/-)-[3H]nicotine and cytisine, anabasine, cotinine and choline selectively inhibited binding at the high affinity site. Several lines of evidence indicate that there is stereospecificity. (+)-Nicotine was more potent than (-)-nicotine in inducing positive cooperativity whereas (-)-nicotine was 80 times more potent than (+)-nicotine in inhibiting binding at the high affinity site. Further, the specificity of the binding sites can be altered by changing the concentration of the buffer which gives additional evidence for the lability of the nicotine binding site. Although the pharmacologic significance of the different binding sites has not been determined, these data taken together indicate that (+/-)-[3H]nicotine binds with specificity to multiple sites in the rat brain P2 preparation with a complexity not addressed heretofore.

The pharmacology of verapamil. V. Tissue distribution of verapamil and norverapamil in rat and dog.

The relative distribution of verapamil and its demethylated metabolite, norverapamil, was studied in rats at intervals after intraperitoneal injection of the parent drug (30 mg/kg). This route of drug administration simulated oral drug dosing, and the highest concentrations of both unchanged drug and metabolite were found in the liver, with lung and kidney containing most of the remainder. The rates of disappearance of verapamil from various organs followed first-order kinetics, and the most rapid elimination occurred from brain and liver. In contrast, verapamil was given intravenously to 3 dogs by a bolus-infusion method to produce sustained steady state plasma concentrations (80, 140, 250 ng/ml) for 1, 2, and 3 h. After systemic administration, the lungs contained almost half the tissue verapamil and, 20% was found in kidney, with the liver accounting for only 17%. Norverapamil was not found in plasma or brain. These studies contrast the pattern of tissue distribution of verapamil after different routes of drug administration. The variable rates of drug elimination from specific tissues may explain the differing durations of the drug's observed effects.

Hemodynamic consequences of combined beta-adrenergic and slow calcium channel blockade in man.

The administration of verapamil to patients receiving beta-adrenergic blocking drugs is reported to produce adverse circulatory reactions, but a systematic investigation of this potential drug interaction has not been performed in man. We administered 40-, 80- and 120-mg doses of verapamil orally to 15 patients with angina pectoris who were receiving high doses of propranolol or metoprolol. Verapamil produced dose-dependent decreases in cardiac performance: with 120 mg, cardiac index decreased by 0.38 l/min/m2, stroke volume index decreased by 2.8 ml/beat/m2 and heart rate decreased by 6 beats/min, associated with increases in pulmonary capillary wedge (2.2 mm Hg) and mean right atrial pressures (1.7 mm Hg) (all p less than 0.01); two patients had marked, but asymptomatic, hypotensive reactions. In contrast, repeat administration of 120-mg doses of verapamil 24--30 hours after withdrawal of beta blockade produced no significant cardiodepressant effects despite significantly higher plasma levels of verapamil than during propranolol therapy (383.1 vs 205.1 ng/ml, p less than 0.01). In conclusion, verapamil produces significant negative inotropic and chronotropic effects in patients treated with beta-adrenergic antagonists; combination therapy should therefore be used with caution in patients with angina pectoris.

Measurement of verapamil concentrations in plasma by gas chromatography and high pressure liquid chromatography.

This study was carried out to compare gas chromatography (GC) and high-pressure liquid chromatography (HPLC) procedures in the measurement of plasma levels of verapamil. Other analytic methods previously reported are not widely available (mass fragmentography) or are subject to interference from drug metabolites (spectrophotofluorometry). A single extraction and derivatization procedure was developed to prepare samples for either GC or HPLC analysis. The GC procedure used a nitrogen-specific detector; the HPLC, a fluorescence detector. In a 1--500 ng/ml range of verapamil concentrations, both methods resulted in good separation of verapamil from a major metabolite, norverapamil, and from compound D517, used as an internal standard. Intraassay variation was similar for both procedures, with only slightly higher interassay variability found for the GC technique. Excellent correlation was found during analysis of the same unknown samples by both methods (r = 0.97; p less than 0.001). Either assay procedure appears satisfactory for use in measurement of verapamil levels in plasma.