Arteether-induced brain injury in Macaca mulatta. I. The precerebellar nuclei: the lateral reticular nuclei, paramedian reticular nuclei, and perihypoglossal nuclei.

Malaria poses a threat across several continents: Eurasia (Asia and parts of Eastern Europe), Africa, Central and South America. Bradley (1991) estimates human exposure at 2,073,000,000 with infection rates at 270,000,000, illnesses at 110,000,000, and deaths at 1,000,000. Significant mortality rates are attributed to infection by the parasite Plasmodium falciparum, with an estimated 90% among African children. A worldwide effort is ongoing to chemically and pharmacologically characterize a class of artemisinin compounds that might be promising antimalarial drugs. The U.S. Army is studying the efficacy and toxicity of several artemisinin semi-synthetic compounds: arteether, artemether, artelinic acid, and artesunate. The World Health Organization and the U.S. Army selected arteether for drug development and possible use in the emergency therapy of acute, severe malaria. Male Rhesus monkeys (Macaca mulatta) were administered different daily doses of arteether, or the vehicle alone (sesame oil), for a period of either 14 days, or 7 days. Neuropathological lesions were found in 14-day arteether treated monkeys in the precerebellar nuclei of the medulla oblongata, namely: (1) the lateral reticular nuclei (subnuclei magnocellularis, parvicellularis, and subtrigeminalis), (2) the paramedian reticular nuclei (subnuclei accessorius, dorsalis, and ventralis), and the perihypoglossal nuclei (n. intercalatus of Staderini, n. of Roller, n. prepositus hypoglossi). The data demonstrate that the simina meduallry precerebellar nuclei have a high degree of vulnerability when arteether is given for 14 days at dose levels between 8mg/kg per day and 24 mg/kg per day. The neurological consequences of this treatment regimen could profoundly impair posture, gait, and autonomic regulation, while eye movement disorders might also be anticipated.

Vaginal Cytology of the Lesser Bandicoot Rat (Bandicota savilei).

The lesser bandicoot rat (Bandicota savilei) is a wild field rat commonly found in Thailand. Vaginal smears were obtained from five colony-born animals twice daily for 28 days and stained with Giemsa stain. The external appearance of the vulva and appearance of the tip of vaginal swabs were recorded. All cells, including nucleated or keratinized epithelial cells and leukocytes, were quantified by percentage. No changes of the external genitalia were observed. The gross appearance of the vaginal swab may distinguish estrus and diestrus. Vaginal swabs contained a white or yellow material during estrus or diestrus, respectively. Changes in vaginal cytology at each stage of the estrous cycle of the lesser bandicoot rat were similar to those of the common laboratory rat. Estrus stages of the five rats occurred every 3 or 4 days. One female was introduced to a male after diestrus and produced a litter of six pups 27 days after pairing.

Arteether: risks of two-week administration in Macaca mulatta.

Male rhesus monkeys (Macaca mulatta) were administered daily doses of the antimalarial drug arteether. The 14-day treated group received either 24 mg/kg/day, 16 mg/kg/day, or 8 mg/kg/day. The seven-day treatment group received either 24 mg/kg/day or 8 mg/kg/day. All control cases in each group received the sesame oil vehicle alone. Neurologic signs were absent for animals in the seven and 14-day treatment groups except for one monkey which showed diffuse piloerection on day 14, and another monkey receiving 24 mg/kg/day for seven days showed mild lethargy after the fourth day. Mild, sporadic anorexia was noted in all animals by day 14, and a single animal showed diffuse piloerection on day 14. Surgical anesthesia preceded killing by exsanguination and was accompanied by perfusion fixation of the central nervous system. Brain sections were cut and then stained for study by light microscopy. Evidence of neuronal pathology, both descriptive and numerical, was collected. The neuroanatomic and neuropathologic findings demonstrated that arteether produced extensive brainstem injury when administered for 14 days. The magnitude of brainstem neurotoxicity was dose-dependent, where injury was greatest at the 24 mg/kg/day dose level, less at the 16 mg/kg/day dose level, and least at the 8 mg/kg/day dose level. Arteether induced multiple systems injury to brainstem nuclei of 1) the reticular formation (cranial and caudal pontine nuclei, and medullary gigantocellular and paragigantocellular nuclei); 2) the vestibular system (medial, descending, superior, and lateral nuclei); and 3) the auditory system (superior olivary nuclear complex and trapezoid nuclear complex). The vestibular nuclei and the reticular formation were most severely injured, with the auditory system affected less. The cranial nerve nuclei (somatic and splanchnic) appeared to escape damage, with the exception of the abducens nerve nucleus. The same brainstem nuclear groups of seven-day treated monkeys appeared normal. The statistical data are concordant with the descriptive data in demonstrating neurotoxic effects. In summary, no neurologic deficits were detected in any of the vehicle control monkeys (14-day and seven-day cases). Monkeys in the 14-day treatment group were free of clinical neurologic signs throughout the first week. At day 14, fine horizontal nystagmus was seen in one monkey, and another monkey exhibited diffuse piloerection. Monkeys in the seven-day treatment group were free of clinical neurologic signs except for one case. This monkey was treated with 24/mg/kg/day of arteether and exhibited lethargy after the fourth day. These indications of dysfunction arose too late to be practical indicators of neurotoxicity.

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)

Proguanil plus sulfamethoxazole is not causally prophylactic in the Macaca mulatta--Plasmodium cynomolgi model.

New drugs for causal prophylaxis of malaria are needed. A proguanil/sulfamethoxazole combination was investigated using a rhesus monkey model (Macaca mulatta infected with Plasmodium cynomolgi) to determine whether causal prophylaxis could be achieved. When a five-day regimen of proguanil (40 mg/kg/day) combined with sulfamethoxazole (100 mg/kg/day) was used, infection of all animals (6 of 6) was observed, with an extended prepatent period (median 40 days). Two control animals became infected on days 9 and 23 following sporozoite inoculation. Plasma concentrations indicated that proguanil and sulfamethoxazole were adequately absorbed and metabolized to cycloguanil and N4-acetylsulfamethoxazole, respectively. Analysis of liver biopsy specimens demonstrated that the drugs were present two days following sporozoite inoculation but were not detectable one week later. Proguanil plus sulfamethoxazole does not eliminate exoerythrocytic-stage parasites in the rhesus monkey--P. cynomolgi model.

Evaluation of WR250417 (a proguanil analog) for causal prophylactic activity in the Plasmodium cynomolgi-Macaca mulatta model.

The Plasmodium cynomolgi-Macaca mulatta model has been used to test the antimalarial activity of new drugs for both radical cure and casual prophylaxis. The proguanil analog WR250417 (also known as PS-15) was evaluated for causal prophylactic activity in rhesus monkeys infected with P. cynomolgi bastianelli. Four monkeys were orally dosed with 40 mg/kg/day of WR250417 over three days (-1, 0, and +1). Sporozoite-induced infection of P. cynomolgi was initiated on day 0 with 1 x 10(6) sporozoites to each monkey. Compound WR250417 extended the prepatent period from an average of 8.5 days for controls (n = 2) to a mean of 18.3 days (range 18-19 days, n = 4) for drug-treated monkeys. Analysis of plasma drug concentrations by high-performance liquid chromatography showed that the monkeys converted the WR250417 to its putative principal active metabolite WR99210 (a dihydrotriazine). These findings demonstrate that WR250417 and its principal metabolite do not prevent primary infection by P. cynomolgi.

A nonhuman primate model for human cerebral malaria: effects of artesunate (qinghaosu derivative) on rhesus monkeys experimentally infected with Plasmodium coatneyi.

We studied the effects of artesunate on rhesus monkeys infected with Plasmodium coatneyi. Sixteen rhesus monkeys were divided in four groups. Group I consisted of three monkeys that were splenectomized and were treated with three doses (loading dose: 3.3 mg/kg, maintenance doses: 1.7 mg/kg) of artesunate, group II consisted of three monkeys that were treated with three doses of artesunate (same as group I), group III consisted of two monkeys that were treated with one dose (3.3 mg/kg) of artesunate, and group IV consisted of five untreated monkeys. Parasitemias of these groups ranged from 13.3% to 19.5% before treatment. Twenty-four hours after administration, the parasitemia was reduced to 2.2% in group I and to < 0.1% in group II; parasitemia was lowered to 10.6% in group III only 3 hr after drug administration. The rate of sequestration in the cerebral microvessels, which was 29.4% in untreated animals, was < 0.1% in groups I and II (24 hr after treatment), and 2.0% in group III (3 hr after treatment). These data clearly indicate that artesunate not only reduced parasitemia, but also reduced the rate of parasitized red blood cell (PRBC) sequestration in cerebral microvessels. In an immunohistologic study, endothelial-leukocyte adhesion molecule-1 (ELAM-1) was not detected in group I after treatment with artesunate, although the presence of CD36, thrombospondin, intercellular adhesion molecule-1, IgG, and C3 in the cerebral microvessels was not altered. This is the first in vivo study to show that artesunate interferes with continued PRBC sequestration in the cerebral microvessels in cerebral malaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Causal prophylactic and radical curative activity of WR182393 (a guanylhydrazone) against Plasmodium cynomolgi in Macaca mulatta.

Primaquine is the only currently available drug effective against persistent tissue stages of relapsing malaria in humans. Causal prophylactic and radical curative properties of WR182393 (a guanylhydrazone) were investigated as part of an effort to evaluate alternatives to primaquine in the rhesus monkey (Macaca mulatta)/Plasmodium cynomolgi test model. The drug was suspended in dimethylsulfoxide for intramuscular (im) injection. A pilot study indicated causal prophylactic activity in a regimen of 40 mg base/kg/day im for three days beginning the day before intravenous challenge with 1 x 10(6) P. cynomolgi sporozoites. Regimens of 31, 10, 3.1, and 0 mg base/kg/day im for three days were then tested in groups of two monkeys given a similar challenge. The two animals given 31 mg base/kg/day remained parasite-free. Average time to parasitemia for the lower dosage groups was 38, 18, and 8 days respectively. Groups of two monkeys with sporozoite-induced P. cynomolgi infections were also treated for seven days with 31, 10, 3.1, and 0 mg base/kg/day im in combination with 10 mg base/kg/day of chloroquine orally. Both monkeys given 31 mg base/kg/day did not relapse. The average time to relapse following treatment was 48, 29, and 8 days, respectively, for the lower dosage groups. Compound WR182393 is the first non-8-aminoquinoline class of drug to exhibit both causal prophylactic and radical curative properties against a relapsing primate, vivax-like malaria.

Sequestration pattern of parasitized erythrocytes in cerebrum, mid-brain, and cerebellum of Plasmodium coatneyi-infected rhesus monkeys (Macaca mulatta).

Six rhesus monkeys (Macaca mulatta) infected with Plasmodium coatneyi were studied for parasitized red blood cell (PRBC) sequestration in microvessels of the brain. The degree of PRBC sequestration is different in the cerebral, mid-brain, and cerebellar microvessels, with sequestration occurring preferentially in the cerebellum. This pattern resembles that of PRBC sequestration in cerebral and cerebellar microvessels in human falciparum malaria. The morphologic appearance of sequestered cells under light and electron microscopy as well as the PRBC sequestration pattern bolsters the contention that the rhesus monkey infected with P. coatneyi is an appropriate primate model for the experimental study of human cerebral malaria.

Application of an enzyme immunoassay for detecting antibodies in sera of Macaca fascicularis naturally exposed to Mycobacterium tuberculosis.

An enzyme immunoassay (EIA) was developed for detecting mycobacterial antibodies in the sera of 22 Macaca fascicularis following a natural outbreak of tuberculosis. EIAs were conducted using four antigens (lysozyme, triton, or deoxycholate extracts of Mycobacterium tuberculosis or a purified protein derivative) and two conjugates (protein A or antihuman). Mycobacterial antibodies were detected in two of two culture-positive monkeys, in nine of ten tuberculin test-suspect monkeys (culture-negative), and in five of ten tuberculin test-negative monkeys (culture-negative). Results indicate EIA may be of practical value in detecting monkeys exposed to M. tuberculosis.

Pharmacokinetics of diazepam intramuscularly administered to rhesus monkeys.

The purpose of this study was to define the pharmacokinetics of diazepam in monkeys following an im injection of 100 micrograms/kg (the minimum effective dose that prevents nerve agent-induced convulsions in pyridostigmine-pretreated, atropine- and 2-PAM-treated monkeys) in order to predict what im dose in humans is needed to prevent nerve agent-induced convulsions. Six rhesus monkeys were administered diazepam in the hind limb. Blood (3 mL) was collected via an indwelling saphenous catheter immediately prior to and 5, 10, 15, 25, 40, 60, 90, 120, 180, and 240 min after diazepam dosing. A contract laboratory, blind to the labeling code, analyzed diazepam serum concentrations by electron-capture gas chromatography and the percentage of unbound diazepam by equilibrium dialysis. The concentration-time data for total (unbound and bound) diazepam individually determined for each animal was best described by a one-compartment open model with first-order absorption and elimination. The average maximum serum concentration (50 ng/mL) was reached in 29 min. The volume of distribution and systemic clearance, assuming 100% bioavailability, were 1.5 L/kg and 19.4 mL/min/kg, respectively. The percentage of diazepam unbound to serum proteins was 4.6% and, therefore, the maximum concentration of free diazepam was 2.3 ng/mL. These results, when compared with human pharmacokinetic studies, allow a means of extrapolating effective monkey anticonvulsant doses to humans on a pharmacokinetic basis.

Pharmacokinetics and pharmacodynamics of oximes in unanesthetized pigs.

The pharmacokinetics and cardiovascular pharmacodynamics of two oximes were studied in unanesthetized pigs. Effects of 2-[(hydroxyimino)methyl]-1-methylpyridinium chloride (pralidoxime chloride; 2-PAM Cl; 50 mumol/kg) were compared with those of 1,1-methylene bis[4(hydroxyiminomethyl) pyridinium] dichloride (methoxime; MMB-4; 100 mumol/kg). Cardiopulmonary parameters were monitored and plasma concentrations of oximes were determined from arterial blood samples taken at intervals over a period of 5 hr postinjection. Plasma concentrations for both oximes were fitted to standard pharmacokinetic models using the computer program PCNONLIN. Average pharmacokinetic parameters were determined for each oxime. Only mild to moderate physiological side effects were detected following intramuscular administration. 2-PAM Cl was more rapidly absorbed and distributed in the blood than MMB-4. Although the latter had a slight lag time to attain detectable levels in the blood, retention time was longer than that of 2-PAM Cl.

Cerebral blood flow-metabolism coupling after administration of soman at nontoxic levels.

The effect of soman, an irreversible organophosphorus cholinesterase inhibitor, on regional cerebral blood flow and glucose utilization were studied with a double-tracer, autoradiographic technique in rats. Soman was given at a subtoxic dose of 55 micrograms/kg SC and variables were measured 45 min later. No changes in arterial blood pressure or signs of toxicity were present in the animals studied. Soman induced a pronounced increase in cerebral blood flow. This change was not accompanied by an increase in cerebral metabolism, with exception of superior colliculi. Brain regions showing the more pronounced (greater than 200% over control) increases in blood flow were motor, sensory and temporal cortex, area 18a of the occipital cortex, claustrum, inferior colliculus and cerebellum. These findings differ from those previously reported for the carbamate cholinesterase inhibitor, physostigmine, and constitute the first demonstration of cerebrovascular effects for an organophosphorus cholinesterase inhibitor, soman, at nonsymptomatic doses.

An attempt to predict anergy in tuberculosis suspect cynomolgus monkeys.

Acid-fast microorganisms were identified from the tuberculous lesions of a male cynomolgus monkey (Macaca fascicularis). Twenty-two other cynomolgus monkeys housed in the same room were presumed exposed to tuberculosis (Mycobacterium spp.). In addition to standard intradermal (ID) tuberculin testing, clinicians attempted to evaluate the immune status of these monkeys in order to identify animals exhibiting false negative (anergy) ID tuberculin tests. Twenty-one of the potentially exposed monkeys were immunized with tetanus toxoid (TT). Tetanus antitoxin (TAT) titers were measured before and after immunization. The delayed cutaneous hypersensitivity (DCH) reaction to TT was evaluated using a commercially available human test panel. Some animals did not exhibit a DCH reaction to TT. At necropsy 1 of the 21 animals exhibited tuberculous lesions, and acid-fast microorganisms were identified on direct smears of lymphatic tissue of a second animal. Although reported to be of value in assessing the cellular immune status of rhesus monkeys (Macaca mulatta), the delayed cutaneous hypersensitivity response to tetanus toxoid was not helpful during this outbreak in identifying cynomolgus monkeys infected with M. tuberculosis, or in interpreting suspect ID tuberculin tests.

Coronavirus-like virions associated with a wasting syndrome in guinea pigs.

An apparent wasting syndrome was observed in newly arriving 3 to 4 week old guinea pigs characterized by anorexia, weight loss, diarrhea, perineal staining and death. Diagnostic efforts to attribute the disease to husbandry, environmental factors or to known guinea pig pathogens were unsuccessful. Clinical signs, enteric histopathological lesions and diagnostic transmission electron microscopy identification of typical coronavirus-like virions in fecal samples were consistent with enteric coronaviral diseases seen in other species.

Effects of physostigmine on the cardiopulmonary system of conscious pigs.

Physostigmine, as a pretreatment candidate for nerve agent poisoning, was examined for cardiopulmonary side effects. Cardiovascular and pulmonary parameters were monitored in unanesthetized domestic pigs which received pulmonary arterial infusion of 5 micrograms/kg/min physostigmine salicylate for 2 hr. A level of 74% inhibition of red blood cell (RBC) acetylcholinesterase (AChE) activity was attained in 45 min, and this level of carbamylation increased only slightly during the remaining infusion period. In addition to this large change in AChE activity, minor changes were observed in hematocrit, heart rate, body temperature, mean aortic pressure, pulmonary arterial wedge pressure, and pulmonary artery pressure. Typically, these parameters showed a trend toward elevated levels. Blood gases, pH, respiratory rate, tidal and minute volume, cardiac output, nonelastic resistance, and dynamic compliance were not significantly different from baseline values. The unanesthetized pig responds to physostigmine in a manner similar to that reported for other species and appears to be a suitable model for evaluating cardiopulmonary effects of cholinesterase inhibitors.