The American Journal of Tropical Medicine and Hygiene - Volume 51, Issue 3, September 1994

Artemisinin (qinghaosu) and several derivatives have been developed and are in use as antimalarial drugs but scant information is available regarding animal or human toxicity. Following a eight-day, multiple-dose, pharmacokinetic study of arteether (AE) (10 mg/kg/day [n = 6] and 20 mg/kg/day [n = 6]) in dogs, all high-dose animals displayed a progressive syndrome of clinical neurologic defects with progressive cardiorespiratory collapse and death in five of six animals. Neurologic findings included gait disturbances, loss of spinal and pain response reflexes, and prominent loss of brain stem and eye reflexes. Animals had prolongation of QT interval corrected for rate (QTc) on electrocardiograms (ECGs) with bizarre ST-T segment changes. Prominent neuropathic lesions were noted to be primarily limited to the pons and medulla. Similar lesions with dose-related severity were noted in eight other dogs studied in a second study with intramuscular (IM) administration of AE in sesame oil during a 28-day, dose-ranging study using 5, 10, 15, and 20 mg/kg/day. Injury, graded by a pathologist blinded to the dose group, showed a dose-related, region-specific injury in all animals that was most pronounced in the pons. Further studies in Sprague-Dawley rats using IM administration of AE and artemether (AM) at a dose of 12.5–50 mg/kg/day for 28 days confirmed the onset of a clinical neurologic syndrome with dose-related changes in body weight, activity, and seizure-like activity, stereotypic movement disorders, and ECG changes. Neuropathologic examination of rat brain sections at five levels from the rostral cerebrum to the caudal medulla showed a dose-related, region-specific pattern of injury characterized by a loss of Nissl substance and hyalinized neuron cell bodies; these changes are congruent with those noted in dogs. No significant differences were noted in the extent, type, or distribution of lesions in brains of rats treated with equivalent doses of AE or AM. We conclude that 1) a dose-related neurologic syndrome associated with movement disturbances, spasticity, and depressed sensorium in dogs and rats occurred after daily IM injections of two artemisinin antimalarial drugs, AE and AM; 2) a prolonged QTc interval was noted as a preterminal clinical finding in dogs and rats treated with high-dose AE; 3) central nervous system neuropathic changes were noted to occur in a dose-related and anatomic-specific manner in both dogs and rats treated with daily IM injections of AE or AM; and 4) the mechanism and etiology for this lesion are not known from this study, but the findings suggest a long-lived toxic drug metabolite.

Wild-caught Anopheles gambiae s. l. and An. funestus were dissected and their midguts were examined for the presence of Plasmodium falciparum oocyst infections. The mean intensity of infection and the prevalence of infected mosquitoes were determined for each sample, with one sample representing the mosquitoes caught in a single house at any given time. The patterns of infection were investigated using the relationships between prevalence, intensity, and variance within samples, and were found to be consistent with laboratory infections. The overall distribution of oocysts is characterized by a mixture of negative binomial distributions with means determined by the infectiousness of the human hosts, and a constant degree of aggregation (k = 0.0767) presumably determined by the development of oocysts within mosquitoes. The prevalence/intensity relationship was treated as a bivariate distribution to ascertain the effect of sample size on the accuracy of estimation, and to allow inference of intensity from prevalence. In mathematical models fitted to the collected data, sample size affected directly the minimum possible prevalence of infection, and the accuracy of both mean and prevalence estimations. Based on minimum possible positive prevalence rates, data from samples of less than 20–25 mosquitoes would provide unacceptable errors in prevalence estimations. However, natural oocyst rates are consistently higher than the minimum prevalence, and it is suggested that any interpretations from samples of less than approximately 40 mosquitoes must be treated with some caution. Such variation in natural samples means that prediction of intensity of infection from prevalence (or vice versa) is extremely inaccurate.

A landscape approach using remote sensing and geographic information system (GIS) technologies was developed to discriminate between villages at high and low risk for malaria transmission, as defined by adult Anopheles albimanus abundance. Satellite data for an area in southern Chiapas, Mexico were digitally processed to generate a map of landscape elements. The GIS processes were used to determine the proportion of mapped landscape elements surrounding 40 villages where An. albimanus abundance data had been collected. The relationships between vector abundance and landscape element proportions were investigated using stepwise discriminant analysis and stepwise linear regression. Both analyses indicated that the most important landscape elements in terms of explaining vector abundance were transitional swamp and unmanaged pasture. Discriminant functions generated for these two elements were able to correctly distinguish between villages with high and low vector abundance, with an overall accuracy of 90%. Regression results found both transitional swamp and unmanaged pasture proportions to be predictive of vector abundance during the mid-to-late wet season. This approach, which integrates remotely sensed data and GIS capabilities to identify villages with high vector-human contact risk, provides a promising tool for malaria surveillance programs that depend on labor-intensive field techniques. This is particularly relevant in areas where the lack of accurate surveillance capabilities may result in no malaria control action when, in fact, directed action is necessary. In general, this landscape approach could be applied to other vector-borne diseases in areas where 1) the landscape elements critical to vector survival are known and 2) these elements can be detected at remote sensing scales.

Gymnophalloides seoi (Digenea: Gymnophallidae), a new human intestinal trematode reported from a Korean woman who complained of epigastric discomfort, has been shown to be highly prevalent among the villagers of a southwestern island of the Republic of Korea. For the detection of human infections, fecal examinations were conducted on the inhabitants of a seashore village, where the first patient with a G. seoi infection had resided. Of 98 inhabitants examined, 70 (71.4%) were infected with various intestinal parasites; among them, G. seoi showed the highest rate of egg positivity 48 of 98 (49.0%). Individual worm burdens of G. seoi, as measured by collection of adult flukes after anthelmintic treatment and purgation, ranged from 106 to 26,373 specimens (average per infected case = 3,326). There was no sex-related difference in the prevalence of G. seoi, and the age distribution of the infected cases showed a relatively even pattern. This study confirms that human infection by G. seoi is not an incidental one and provides the first evidence of its high prevalence on this Korean island.

The purposes of this study were to determine if hantaviruses were present in the Great Lakes port areas of Wisconsin and Minnesota and if so, to identify which virus and which rodent host species were involved. Rodents were trapped in Duluth, Minnesota, Superior, Green Bay, and Milwaukee, Wisconsin, all ports of call for international maritime shipping. A total of 675 wild rodents were captured and tested, including 310 meadow voles (Microtus pennsylvanicus), 173 Norway rats (Rattus norvegicus), 179 Peromyscus spp., (including white footed mice [P. leucopus] and deer mice [P. maniculatus gracilis and P. maniculatus bairdii]), and 13 house mice (Mus musculus). Twenty percent of the rats, 17% of the meadow voles, 8% of the house mice, and 3% of the Peromyscus spp. had antibody to a hantavirus by immunofluorescent antibody assay (IFA). By the plaque-reduction neutralization test (PRNT), nine of 36 meadow voles, one of 4 P. leucopus, and one of 34 rats had hantavirus antibody, with the highest titers to Prospect Hill (PH) virus. All of the PRNT-seropositive individuals were from the twin cities of Superior and Duluth. Hantavirus antigen was detected in lung tissue by IFA in M. pennsylvanicus and Peromyscus spp., but not in rats. Two hantaviruses, designated SD-1 and SD-2, were isolated from M. pennsylvanicus captured in Duluth and found to be very similar to prototype PH virus by cross-IFA and cross-PRNT. Virus isolation attempts were unsuccessful from tissues of the Peromyscus spp. and the rats.

A colony of rosy eye mutants of Aedes aegypti was established. This strain was refractory to Chikungunya virus by oral route of infection when compared with the wild-type parent strain. The refractoriness of this strain seems to be due to a mesentronal barrier, since both the mosquito strains supported the multiplication of virus after intrathoracic inoculation. The rosy eye strain was also found to be refractory to Sagiyama virus (Alphaviridae: Getah virus subtype) when compared with wild-type parent strain, but no such difference in the oral susceptibility was found with dengue-2 (Flaviviridae) virus. The rosy eye mutant appears to be closely linked to the gene(s) for refractoriness to alpha viruses and may be useful in future studies in understanding the genetic basis of vector competence of Ae. aegypti to arboviruses.

Parasitic protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) are generally thought to multiply by binary fission; however, data from quantitative microspectrophotometry indicate that nuclear fusion or sexual reproduction takes place in the intracellular amastigote form. Among several different Leishmania species, the mean ± SD nuclear DNA content of all promastigotes (extracellular form) and of some amastigotes (intracellular form) in macrophages was 0.098 ± 0.032 relative units; in contrast, other amastigotes within the same macrophage had a mean ± SD nuclear DNA content of 0.219 ± 0.050. The latter population of amastigotes are apparently produced when the nuclei of a pair of 0.098 amastigotes fuse. These 0.219 amastigotes later go through what is probably the typical meiotic cycle to reform the 0.098 condition we observed among promastigotes. The demonstration of sexual reproduction in Leishmania has important implications for the future direction of research on this medically important parasite.

A highly sensitive, rapid and simple method to detect human malaria in blood samples was developed. Malaria parasite DNA in blood from a fingerprick was directly amplified by the polymerase chain reaction (PCR) using two sets of primers to yield a 206-basepair (bp) product for Plasmodium falciparum and a 183-bp product for P. vivax. Both were easily visualized in an ethidium bromide-stained agarose gel, allowing identification of the two human malaria species in a single amplification reaction. As little as a one P. falciparum and/or P. vivax parasite per microliter of blood was detectable by this method, a sensitivity superior to that of thick blood film microscopy. The total time required for diagnosis of 48 blood samples, starting from fingerprick blood collection, was approximately 4 hr. When compared with microscopic examination by an expert microscopist, results showed a sensitivity of 89% for P. falciparum and 91% for P. vivax and an overall specificity of 94%. Six infected blood samples classified by microscopy as single species were diagnosed by the PCR method as being mixed P. falciparum and P. vivax infections. The high sensitivity, rapidity, and simplicity of the method should make it attractive for a large-scale epidemiology study, follow-up of drug treatment, and immunization trials.

There is need for sensitive, rapid, species-specific diagnosis of Brugia filarial parasites because traditional methods are tedious and time-consuming, with little guarantee of species specificity. A polymerase chain reaction (PCR)-based assay was developed using the Hha I family of highly repeated DNA sequences from Brugia. The assay was tested on 124 human blood samples collected in a field study in Indonesia. These included 66 microfilaria-positive samples from patients in an area endemic for Brugia, 30 from healthy individuals from the same endemic area, and 28 from healthy individuals from a nonendemic area. Twenty-eight blood samples collected in a village in French Polynesia endemic for Wuchereria bancrofti, but not B. malayi, were also tested. The blood samples were screened using the traditional blood smear and membrane filtration methods, which served as the gold standards to which the PCR assay was compared. The samples were digested with proteinase K, extracted with phenol and chloroform, and dialyzed. A fraction of the dialyzed product was used in PCRs using Hha I-specific primers. The PCR assay correctly identified all of the microfilaria-positive samples as PCR positive and all of the nonendemic samples as PCR negative. Additionally, 26 of 30 samples from healthy individuals in the endemic area were also identified as PCR negative, while four were PCR positive. It is likely that these four individuals had very low-level or cryptic infections, and that the PCR assay detected circulating DNA released from dead filariae. The results indicate that the Hha I PCR detection system is rapid, species-specific, and sensitive.

Using an anti-oocyst wall monoclonal antibody-based immunofluorescence assay, the presence of Cryptosporidium parvum was evaluated in children with diarrhea from rural areas (selected from a door-to-door community survey) and from urban areas (patients attending hospitals) in the State of Puebla, Mexico. Prevalences of 9.4% in a rural population (n = 85) and 29.6% in a hospital-based urban population (n = 81). There was no consistent correlation between water source and other environmental data and the presence of C. parvum in stools. It is concluded that C. parvum may be an important pathogen associated with diarrhea in this Mexican state.

To determine the occurrence and factors associated with Cryptosporidium among persons with acquired immunodeficiency syndrome (AIDS) in Los Angeles County, data were analyzed from the AIDS surveillance registry for the 10-year period 1983–1992. Among 16,953 persons with AIDS, a total of 638 (3.8%) cryptosporidiosis cases were reported during the study period. The prevalence of cryptosporidiosis was higher in persons whose suspected human immunodeficiency virus (HIV) exposure category was through sexual contact (3.9%) than among persons in other HIV exposure categories (2.6%; P < 0.01) and in immigrants from Mexico (5.2%) than in American born patients (3.8%; P < 0.01). Blacks (2.7%) were less likely than whites (4.1%) and Latinos (4.2%) to be reported with cryptosporidiosis (P < 0.001). A temporal trend was observed from 1983 to 1986 when the prevalence decreased from 6.7% to 3.6% (P < 0.001, by chi-square test for trend). After controlling for confounding variables by stratified analysis, persons whose HIV exposure was sexual (adjusted odds ratio [OR] = 1.7, 95% confidence interval [CI] 1.3, 2.4, P < 0.01) and immigrants from Mexico (adjusted OR = 1.6, 95% CI 1.2, 2.1, P < 0.01) were more likely to have cryptosporidiosis. The negative association with black race remained significant (adjusted OR = 0.7, 95% CI 0.57, 0.96, P = 0.02). The prevalence of cryptosporidiosis decreased with age in gay and bisexual males (Mantel-Haenszel test for trend, P < 0.01) but not among female and heterosexual male cases. The temporal trend of decreasing prevalence observed was a result of a decrease in cryptosporidiosis prevalence among gay and bisexual men with AIDS from 1983 to 1986 (P < 0.01, by Mantel-Haenszel test for trend), which corresponded to a time of modified sexual behavior in this group. These data suggest the existence of modes of Cryptosporidium infection in persons with AIDS, including sexual transmission among gay and bisexual men and the occurrence of travel-related infections. Such information may be of value in recommending strategies for preventing Cryptosporidium infection in HIV-infected persons.

A captive-born rhesus monkey (Macaca mulatta) experimentally infected with simian immunodeficiency virus developed neurologic abnormalities approximately seven months postinoculation. A chronic necrotizing encephalomyelitis with intralesional protozoal schizonts was diagnosed histologically. The protozoa was identified as Sarcocystis neurona based on its morphologic characteristics by light and electron microscopic examination, the developmental stages of the schizonts, and positive staining with antisera against Sarcocystis cruzi by immunocytochemical techniques. Although S. neurona may be confused with Toxoplasma gondii by light microscopy, the former lacks rhoptries, is in direct contact with the host cell cytoplasm, and divides by endopolygeny. Sarcocystis neurona has recently been identified as an etiologic agent of encephalomyelitis in horses, raccoons, and mink.

Strongyloides stercoralis ordinarily matures in the human proximal small intestine and embeds in the small intestine mucosal wall where eggs are produced. Although Strongyloides may infect many sites in hyperinfection syndromes, reports of gastric involvement are rare. We report a patient taking prednisone and an H2 blocker who developed hyperinfection syndrome, with mucosal Strongyloides larvae seen in a gastric biopsy.

The zoonotic hookworm, Ancylostoma caninum, probably induces human eosinophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in other parasites, cysteine proteinases are involved in pathogenesis. We studied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activity using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity was observed against the cathepsins L and B-specific substrate Z-phe-arg-AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against gelatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in adult extracts was enhanced up to 15-fold by the reducing agent dithiothreitol (DTT), but was totally blocked by specific inhibitors of cysteine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-CHN2, E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gelatinolytic activity in ES antigens detected using substrate gels was enhanced by the addition of reducing agents and inhibited by Z-phe-ala-CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity in ES antigens was active over a wide pH range (pH 5–9). Similar cysteine proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the substrate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydrolysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cysteine proteinase activity. Furthermore, since the specific activity of Z-phe-arg-AMC-cleaving enzyme was up to five-fold higher in ES antigens than in soluble extracts of parasites, the cysteine proteinase is actively secreted by adult hookworms. This is the first demonstration of cysteine proteinases from hookworms and enlarges the range of potential allergens that may induce human eosinophilic enteritis.

Antigen detection assays serve as a useful adjunct to blood examinations for studies of filariasis, in terms of the diagnostic and epidemiologic information provided. We examined the utility of the Og4C3 antigen detection enzyme-linked immunosorbent assay for field studies and analyzed the distribution of Wuchereria bancrofti antigenemia in a Haitian population. Using serum samples collected following venipuncture, antigenemia levels were correlated with microfilaremia (P < 0.001). The microfilariae had a pronounced nocturnal periodicity while the sensitivity of the antigen assay was the same whether serum samples were collected during the day or at night. To determine whether the Og4C3 assay could be used in conjunction with fingerprick blood examinations, nocturnal blood surveys were conducted. Of 419 persons surveyed, 207 (49.4%) were antigen-positive with the Og4C3 assay. Serum specimens from all 121 microfilaremic individuals were antigen positive (100% sensitivity). The age prevalence of antigenemia increased from 24.5% for 1–5-year-old children to 70% for persons greater than 50 years of age. These results demonstrate that the Og4C3 assay is a sensitive tool for the detection of infection and raise questions about the expression of protective immunity in populations exposed to infection.

To assess natural immunity against the circumsporozoite (CS) protein and the synthetic vaccine SPf66, immunologic studies were carried out in a highly endemic malarious area of Papua New Guinea. Antibody prevalence, antibody titers, and T cell proliferation against both antigens were measured in 214 adults. Immunologic data were analyzed with respect to longitudinal malariologic and morbidity data. Evidence of genetic traits such as glucose-6-phosphate dehydrogenase deficiency and ovalocytosis was analyzed. Antibody prevalence was high, with 79% and 84% for CS protein and SPf66, respectively, while T cell proliferation was infrequent and low, with 14% and 12% responders, respectively. Anti-CS protein antibodies increased with age but showed no association to malaria indices or morbidity. No protective value was observed with T cell responses or with humoral response to SPf66. These results provide a first description of naturally developed immunity against SPf66 and suggest further studies in to fully understand the mechanism of immunity against this antigen.

We have shown in a previous study that immunization of a rhesus monkey (Macaca mulatta) with inactivated liver stages of the simian malaria parasite Plasmodium cynomolgi (B strain) produced high antibody titers against sporozoites, liver stages, and blood stages of P. cynomolgi. In the present study, we demonstrate that these anti-P. cynomolgi immune sera recognized P. vivax (Salvador I) antigens. In an indirect immunofluorescence assay, both postimmunization and postchallenge sera reacted with antigens of sporozoite, liver-, and blood-stage parasites. In Western blot analysis, postimmunization sera recognized four bands of 97, 93, 70, and 65 kD in sporozoite antigens; postchallenge sera further recognized three doublet (set of two) bands of 86–90, 73–78, and 44–52 kD. When blood-stage extracts were used as antigens, five bands of 118, 105, 76, 68, and 47 kD reacted with postimmunization sera; two doublet bands of 81–87 and 49–52 kD further reacted with postchallenge sera. None of these sera reacted with asexual blood-stage extracts of P. falciparum and P. malariae, or with a recombinant circumsporozoite protein of P. vivax.

To explain the observation that acute Plasmodium falciparum malaria is associated with a transient inability of peripheral blood cells to respond to antigenic stimulation in vitro, we have postulated the disease-induced reallocation of peripheral lymphocytes, possibly by adhesion to inflamed endothelium. We measured plasma levels of soluble markers of endothelial inflammation and T cell activation in 32 patients suffering from acute, uncomplicated P. falciparum malaria, as well as in 10 healthy, aparasitemic control donors. All donors were residents of a malaria-endemic area of Eastern State Sudan. In addition, we measured the T cell surface expression of the interleukin-2 receptor (CD25) and the lymphocyte function-associated antigen (LFA-1; CD11a/CD18). We found that the plasma levels of all inflammation and activation markers were significantly increased in the malaria patients compared with the control donors. In addition, we found a disease-induced depletion of T cells with high expression of the LFA-1 antigen, particularly in the CD4+ subset. The results obtained provide further support for the hypothesis of T cell reallocation to inflamed endothelium in acute P. falciparum malaria.

Even with the availability of highly effective drugs that can prevent Pneumocystis carinii pneumonia (PCP), this infection continues to be the greatest single defining illness of acquired immunodeficiency syndrome (AIDS). Despite significant progress over the last decade in our knowledge about the diagnosis and treatment of this infection, the fact that so much of the basic biology and epidemiology of P. carinii remains unknown continues to be a source of frustration to scientists working with this parasite. Pneumocystis carinii Pneumonia, edited by Peter D. Walzer, M.D., Chief of the Infectious Diseases Section of the Cincinnati Veterans Affairs Medical Center, documents both the recent progress in our knowledge as well as the continued frustration (based principally on our inability to culture the organism) that this new knowledge has come so haltingly and at such high cost.

The volume contains 31 chapters written by leading authorities on PCP.

It is divided into six parts: basic biology, epidemiology, pathophysiology, clinical features, diagnosis, and treatment and prevention.