Cultivation in vitro of the quartan malaria parasite Plasmodium inui.

The simian guartan malaria parasite Plasmodium inui (OS strain) was cultured in a continuous flow system with rhesus monkey erythrocytes and RPMI 1640nmedium supplemented with Hepes buffer and rhesus serum. Over a 10-week period, the growth of the parasite permitted a 61,000-fold cumulative dilution of the original inoculum. After 5 weeks in culture, the parasites were still infective to the monkey Saimiri sciureus and to Anopheles freeborni mosquitoes.

Plasmodium falciparum gametocytes from culture in vitro develop to sporozoites that are infectious to primates.

Gametocytes of two strains of the human malaria parasite Plasmodium falciparum have been produced in high density by means of a continuous-flow cultivation system. The gametocytes of these two strains infected a mean of 36 percent and 71 percent, respectively, of Anopheles freeborni mosquitoes that fed on a suspension of red blood cells containing the culture gametocytes. Sporozoites harvested from the infected mosquito salivary glands were infective to the chimpanzee (Pan troglodytes) and the owl monkey (Aotus trivirgatus).

Cultivation in vitro of the vivax-type malaria parasite Plasmodium cynomolgi.

The vivax-type simian malaria parasite Plasmodium cynomologi was cultured in vitro by both the candle jar method and the continuous flow technique, with rhesus monkey erythrocytes and RPMI 1640 medium supplemented with Hepes buffer and human serum. After 6 weeks in culture, the growth of the parasite had permitted a 5 X 10(6) cumulative dilution of the original inoculum. Cultured parasites remained infective to rhesus monkeys and exhibited a reversible decrease in the ameboid behavior of their trophozoites.

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

Blood transfusion practices and blood-banking services in a Kenyan hospital.

OBJECTIVES: To identify ways to improve the operation of blood-screening programs and to decrease the inappropriate use of blood by evaluating blood-transfusion practices and blood-banking services in a Kenyan hospital. DESIGN: Prospective cohort. SETTING: The study was conducted in a rural district hospital in western Kenya between September 1990 and July 1991. METHODS: We collected data on all transfusion requests (blood donation, grouping, HIV screening) and blood recipients (age, sex, diagnosis, and for a 3-month period on the pediatric, maternity, and female wards, admission hemoglobin and outcome). RESULTS: During the 11-month study period, 799 patients received 927 transfusions: 67% were children < 15 years of age, 27% were adult women and 6% were adult men. Transfusions were often delayed due to reliance on patient-recruited donors. Patients who received blood donated on or after the date of request waited longer for transfusion (median, 3 days) than patients who received blood that had been banked and screened before the request (median, 1 day). Patient-recruited donors had a higher HIV-seropositivity rate than volunteer donors (13.4 and 4.6%, respectively; chi 2 test, P < 0.001). Overall, 47% of pediatric transfusions were classified as inappropriate: 23% did not meet the criteria of having hemoglobin < 5.0 g/dl and clinical evidence of respiratory distress, and 27% were transfused 2 or more days after requested. Among adults, 68% received one unit of blood or less. CONCLUSIONS: Improved laboratory services, reduction of unnecessary transfusions, and increased recruitment of volunteer donors are critical for improving the appropriate and timely use of blood and reducing transfusion-associated HIV transmission.

PIP: Between September 1990 and July 1991, health workers and/or laboratory personnel at Siaya District Hospital in rural western Kenya (about 60 km northwest of Kisumu) gathered data on 799 patients who received 927 blood transfusions, including blood donation, grouping, and HIV screening. Most blood recipients were children (under 15 years old). Only 6% of all recipients were men. Just 30% of transfusions were performed the day of request. Blood donors recruited when it was most needed for survival. Their blood tended to be available 3 days after the request. The volunteer donated blood tended to be available for transfusion the day of request, however, because it had already been banked and screened. Patient-recruited donors were more likely to be HIV infected than volunteer donors (13.4% vs. 4.6%; relative risk = 2.91; p .001). 47% of the pediatric transfusions should not have taken place because 23% of these children did not suffer respiratory distress and their hemoglobin levels were greater than t gm/dl and because 27% received the transfusion 2 days after the day of request. 90% of all adult transfusions were inappropriate (i.e., transfusion of no more than 1 unit of blood or received the transfusion 2 days after the day of request). 30% of blood units that had been banked and screened at the time of request were not transfused until at least 2 days after request. These findings identified those areas which must be targeted to improve the appropriate and timely use of blood and reducing transfusion-induced HIV transmission: reduction of inappropriate transfusions, increased recruitment of volunteer donors, and improved laboratory services.

Longitudinal evaluation of severely anemic children in Kenya: the effect of transfusion on mortality and hematologic recovery.

OBJECTIVE: To determine the effect of transfusion on hematologic recovery and mortality among severely anemic children during and after hospitalization in rural Kenya. DESIGN: Prospective cohort. METHODS: We collected clinical and laboratory information on all severely anemic children (hemoglobin < 5.0 g/dl) and a 33% sample of children with hemoglobin < or = 5.0 g/dl who were admitted to the pediatric ward of a rural Kenyan hospital during a 6 month study period. Children were followed during hospitalization and at 4 and 8 weeks after admission. RESULTS: Overall, 303 (25%) of the 1223 hospitalized children had hemoglobin < 5.0 g/dl, 30% of whom died during the study period. Severely anemic children who were transfused had a higher mean hemoglobin level at discharge (9.0 g/dl) than non-transfused children (5.8 g/dl, P < 0.001) and maintained a higher mean hemoglobin during the 8-week follow-up period. However, the presence of malaria parasitemia on follow-up negated the benefit of transfusion on hematologic recovery at both 4- and 8-week visits (longitudinal linear model, least square means, P > 0.05). Transfusion was associated with improved survival among children with respiratory distress who received transfusions within the first 2 days of hospitalization. CONCLUSIONS: The use of transfusion can be improved by targeting use of blood to severely anemic children with cardiorespiratory compromise, improving immediate availability of blood, and treating severely anemic children with effective antimalarial therapy.

PIP: The effect of blood transfusion on hematologic recovery and mortality both during and after hospitalization was investigated in a survey of children admitted to Siaya District Hospital (Kenya) in a 6-month period in 1991 with hemoglobin under 5.0 g/dl (n = 303) or 5.0 g/dl and above (n = 303). Children with hemoglobin under 5.0 g/dl (severe anemia) were younger and more likely to have malaria parasitemia and respiratory compromise than controls. 88 severely anemic children (30%) died during the study period. Severely anemic children who were transfused had a higher mean hemoglobin level at discharge (9.0 g/dl) than nontransfused children (5.8 g/dl) and maintained a higher mean hemoglobin in the 8-week post-discharge follow-up period. 15% of transfused and 17% of nontransfused children died after hospital discharge. Transfusion was associated with significantly improved survival among children with respiratory distress who were transfused within 2 days of hospital admission. However, the presence of malaria or parasitemia at follow-up negated the benefit of transfusion on hematologic recovery. These findings suggest that the effectiveness of transfusion can be enhanced by targeting severely anemic children with cardiorespiratory compromise, improving immediate access to blood, and effective antimalarial therapy. In addition, more information is needed on the causes of death among anemic children and the prevention of severe anemia.

Decreased regional brain metabolism after ect.

OBJECTIVE: The antidepressant action of ECT may be related to its anticonvulsant properties. Positron emission tomography (PET) studies of regional cerebral metabolic rate for glucose were used to test this hypothesis. METHOD: Ten patients with major depression were studied with PET before and approximately 5 days after a course of bilateral ECT. Statistical parametric mapping was used to identify regions of decreased cerebral glucose metabolism. RESULTS: Widespread regions of decreased regional cerebral glucose metabolism were identified after ECT, especially in the frontal and parietal cortex, anterior and posterior cingulate gyrus, and left temporal cortex. A region-of-interest analysis similarly indicated post-ECT reductions in regional cerebral glucose metabolism. CONCLUSIONS: ECT reduces neuronal activity in selected cortical regions, a potential anticonvulsant and antidepressant effect.

A review of five years' experience with rubella vaccine in the United States.

National morbidity figures show a decline in reported rubella and congenital rubella syndrome since 1969, concurrent with widespread use of rubella vaccine. In addition, no nationwide outbreak, such as the 1963-1964 epidemic, has occurred, though on the basis of long-term secular trends, one would be expected between 1970 and 1974. Recent rubella outbreaks have occurred in unimmunized students in high schools and universities, and there appears to have been a slight upward shift in the age-specific incidence of rubella in the United States since the beginning of widespread immunization. Currently available vaccines have provided durable protection to date, and, although reinfection is known to occur following vaccination, it has not proven a risk to the pregnant woman. There is a small but significant incidence of adverse reactions and a potential risk to the woman who is vaccinated during pregnancy. These data indicate that rubella vaccines are safe and effective. They also imply that rubella vaccines, as they are currently applied, have been successful in reducing the morbidity of congenital rubella syndrome, although continued surveillance will be necessary to confirm this trend.

Intracoronary thallium-201 assessment of thrombolysis in acute myocardial infarction. Validation of the method of imaging before and after therapy.

In order to study acute changes in perfusion with intracoronary thrombolytic therapy, we have used ten times the pretherapy intracoronary thallium-201 dose for the posttherapy study. Because of the larger posttherapy dose, the posttherapy images had ten times as many counts as the pretherapy images. Since the change in image quality between the pretherapy and posttherapy studies might affect interpretation, we studied the effect of image statistics on interpretation of perfusion scintigraphy. The pretherapy and posttherapy images were scored on a four-point scale in five segments on each of three views. In 31 patients, Poisson-distributed pseudorandom noise was added to the posttherapy study in order to match the statistical accuracy of the pretherapy study. A blinded interpretation of the pretherapy and posttherapy noise-added images was performed in the same way as the initial unblinded interpretation. The mean difference between the unblinded pretherapy and posttherapy scores (the improvement in thallium distribution with therapy) was 2.5+/-0.8 (standard error) compared with the difference between the blinded pretherapy and posttherapy noise-added scores which was 2.6+/-1.0. The correlation between readings of similar pairs of data was higher than the correlation between pretherapy and posttherapy studies. Thus, the difference in statistic quality of the pretherapy and posttherapy studies did not affect the interpretation of these studies. Therefore, our evaluation of pretherapy and posttherapy studies using a ten-fold increase in thallium-201 dosage is valid.

Malaria. Principles of prevention and treatment.

In the United States, effective malaria prevention strategies for short-term travelers are available. Monitoring trends in imported malaria and continued evaluation of the effectiveness and chemoprophylaxis will allow prevention recommendations to evolve as the risk of infection and effectiveness of antimalarial drugs change. Our challenge is to increase the number of prospective travelers receiving pre-travel advice, to disseminate this information to health care providers, and to improve the quality of the advice given. The early recognition of Plasmodium infection and the institution of prompt and effective treatment will reduce morbidity and mortality from malaria in this country.

Seroepidemiological studies of malaria in pregnant women and newborns from coastal El Salvador.

A cohort of 113 women and their newborns from the coastal area of El Salvador were studied longitudinally to estimate malaria incidence and indirect fluorescent antibody (IFA) response to malaria infection. The district in which the study was conducted had an estimated annual parasite index of 600/1,000 inhabitants, and all malaria infections were treated immediately with a 4-aminoquinoline. In the third trimester of pregnancy, the IFA response to Plasmodium falciparum was significantly depressed. As a result of antimalarial therapy and depressed immune responsiveness, 49% (P. vivax) and 53% (P. falciparum) of the pregnant subjects had a malaria IFA titer less than 1:20 at the time of delivery. Malaria IFA crossed the placenta to the fetus with a step-down of approximately a 4-fold dilution, except for the step-up noted in the P. falciparum titer for 17 of 116 newborns. Due to the overall low prevalence and intensity of maternal IFA, a titer of at least 1:20 was passed to only 23% (P. vivax) and 45% (P. falciparum) of newborns. Passively-acquired malaria IFA degraded with a half-life estimated between 43 and 52 days. During follow-up of infants to 6 months of age, no protection from malaria resulting from passively-acquired antibody could be demonstrated. Because of the limited transplacental immunization of these newborns with antimalarial antibody, it appears that passive immunity can exert little effect on the incidence of infant malaria in coastal El Salvador.

Clinical and laboratory characterization of a chloroquine-resistant Plasmodium falciparum strain acquired in the Central African Republic.

This case of chloroquine prophylaxis failure occurred in the Central African Republic, a country heretofore unaffected by chloroquine resistance. The clinical findings and chloroquine blood levels and blood smears confirmed prophylaxis failure. In vitro susceptibility testing demonstrated the parasite to be 3- to 4-fold more resistant than a susceptible reference clone.

Developing effective strategies for malaria prevention programs for pregnant African women.

The control of malaria in pregnant African women, one of several child survival strategies applied through antenatal care, has been particularly challenging. Prevention and control recommendations for typical areas of high Plasmodium falciparum transmission have promoted the use of antimalarial chemoprophylaxis to prevent placental infection. Persistently low program coverage coupled with diminishing intervention effectiveness have forced a re-evaluation of the relative importance of malaria in pregnancy. The Mangochi Malaria Research Project (MMRP), a prospective evaluation of malaria prevention in pregnant women in rural Malawi conducted during 1987-1990, contributed to establishing new criteria for policy and program development for malaria prevention in pregnancy. The principle findings of the MMRP include: 1) populations at risk of the adverse consequences of malaria in pregnancy include women with low parity, women infected with human immunodeficiency virus, pregnancy during the high malaria transmission season, and the use of a malaria drug that is suboptimally efficacious; 2) the estimated maximum benefits of an antimalarial intervention that clears placental and umbilical cord parasitemia are a 5-12% reduction of low birth weight (LBW), an approximately 35% reduction in the risk of LBW for risks that are actually preventable once a woman has become pregnant (e.g., risks such as infectious disease or poor nutrition during gestation), and a 3-5% reduction in the rate of infant mortality; 3) the intervention must be capable of rendering the woman malaria parasite free, including clearance of parasites from the placental vascular space and umbilical cord blood; 4) other diseases adversely affect pregnancy outcome and, while the control of malaria in pregnancy may not warrant independent programming, if coupled with prevention programs to provide a range of antenatal services, the incremental costs of malaria control may prove to be highly cost-effective; and 5) the choice of a regimen must balance intervention efficacy with safety, availability, affordability, and simplicity of delivery, and several antimalarials may meet these criteria. The Malawi Ministry of Health has modified its malaria prevention in pregnancy recommendations and now faces the challenge of effective programming to improve child survival.

PIP: During 1987-90, a prospective evaluation of malaria prevention in pregnant women in rural Malawi, the Mangochi Malaria Research Project, was conducted. It aimed to address systematically the evolving obstacles to effective program strategies. The findings contribute to the establishment of new criteria for decision-making in policy and program development for malaria prevention and control in pregnancy. The project resulted in five key lessons learned. Populations at risk of the adverse effects of malaria during pregnancy are low-parity women, HIV-infected women, women pregnant during the high malaria transmission season, and pregnant women using a less effective malaria drug. The estimated maximum benefits of an antimalarial intervention include a 5-12% reduction in low birth weight (LBW), an approximate 35% reduction in the risk of LBW for risks that are preventable once a woman has conceived (i.e., infectious disease or poor nutrition during pregnancy), and a 3-5% reduction in infant mortality. The antimalarial intervention must be able to make the pregnant women malaria-parasite free and to effect clearance of parasites from the placental vascular space and umbilical cord blood. Since other diseases also adversely affect pregnancy outcome, the control of malaria should be integrated with prevention programs to provide a range of prenatal services. When choosing a regimen, the health provider must balance the regimen's efficacy with safety, availability, affordability, and ease of delivery. Several antimalarial regimens appear to meet these criteria. Based on the findings of the project, the Malawi Ministry of Health has changed its recommendations for malaria prevention in pregnancy.

Evaluation of amodiaquine treatment of chloroquine-resistant Plasmodium falciparum malaria on Zanzibar, 1982.

Amodiaquine, a 4-aminoquinoline which has been shown to be effective in treating infections with chloroquine-resistant strains of Plasmodium falciparum, was evaluated against chloroquine-resistant infections in children in Zanzibar, Tanzania, during July 1982. A 25-mg base/kg dosage of amodiaquine produced parasite clearance in 34 of 38 (89%) children in a mean of 2.8 days. When followed for 28 days, 15 of 38 (39%) children were completely cured of their infection as judged by the absence of renewed parasitemia. The parasite clearance rates produced by amodiaquine were significantly higher than those observed in a comparison group of children treated with 25 mg base/kg chloroquine. There was, however, no difference in the cure rates in the chloroquine and amodiaquine groups. Despite the enhanced parasite clearance rate, amodiaquine is not sufficiently more effective against Zanzibari strains of P. falciparum to replace chloroquine. Other alternative drugs must be evaluated to define the optimal malaria therapy regimen on Zanzibar.

The serological response to primary malaria infection in urban Ghanaian infants.

Thirty-one (31) newborn infants living in Accra, Ghana, were visited monthly for the first 15 months of life to determine their serologic response to primary malaria infection. Only 10 episodes of seroconversion were observed, the earliest occurring at the 5th month and at a time when maternal-acquired antibodies were absent. Following seroconversion, antibody titers peaked at the 1st month but were generally of low titer (mean geometric titers < 1:80) and declined to undetectable levels within a few months. The majority of the seroconverting infants had no symptoms of illness although in three of the 10 episodes splenic enlargement was noted. This study suggests that symptoms of malaria infection in infancy are often minimal, but that the moderation of symptoms is due to factors other than maternally transmitted antimalarial antibodies. Additionally, malaria appears to be much less common than expected in this urban area of West Africa.

Early formation of the nodule in Guatemalan onchocerciasis.

Thirty-eight nodules containing adults of Onchocerca volvulus were removed from 36 patients who had no detectable microfilariae in skin snips. Worms were digested from nodules in collagenase solution, maintained alive in vitro, and the number, sex, and state of fecundity were recorded. A total of 48 female and 8 male worms were recovered; 39 females were in the nodules without the presence of a male. Eleven females (22.9%) had microfilariae in utero or produced microfilariae in vitro; seven of these were found together with males in the nodules while four were not. No nodules were found around male worms unless a female was also present. These observations indicate that the nodule forms only around female worms and that mating probably occurs before or early during nodule formation. Furthermore, the production of microfilariae by the the female is not essential for nodule formation since many nodules contained non-fecund, living females.

Plasmodium inui and Babesia microti infections in the squirrel monkey, Saimiri sciureus.

The course of Plasmodium inui and Babesia microti infections was studied in seven splenectomized squirrel monkeys (Saimiri sciureus) of Guyanan or Bolivian origin. Three of the monkeys were infected with P. inui either by the inoculations of parasitized blood or by the bite of infected mosquitoes. The remaining four monkeys were infected by the inoculation of parasitized blood, containing P. inui and B. microti in three and with B. microti only in one. The infection in all seven animals was severe, terminating fatally.

Serologic studies on onchocerciasis in Guatemala using fixed-tissue sections of adult Onchocerca volvulus.

Sera from 458 residents of a coffee plantation in an area of Guatemala endemic for onchocerciasis were examined for the presence of antibodies to Onchocerca volvulus using a fluorescent antibody (IFA) test with sections of adult worms as the antigen. Antibody response increased with age of the residents and microfilarial density. A total of 70.0% of the residents were serologically positive and 65.1% were positive for microfilariae. Males had higher microfilarial densities and higher IFA responses. Sixty-three individuals were positive for microfilariae and IFA-negative indicating a lack of sensitivity on the part of the test antigen. Seventy-four individuals were negative for microfilariae and IFA-positive, suggesting the possible usefulness of the test in the determination of present or past infection in the absence of microfilariae.

Evaluation of sporontocidal compounds using Plasmodium falciparum gametocytes produced in vitro.

A test system that uses infective gametocytes from in vitro cultures was developed for evaluating the sporontocidal activity of antimalarial compounds. In evaluating the system, pyrimethamine and cycloguanil (dihydrofolate reductase inhibitors) and primaquine (8-aminoquinoline) were tested against pyrimethamine-sensitive and pyrimethamine-resistant strains of Plasmodium falciparum. The drugs were administered to Anopheles either in a blood meal containing infective gametocytes or in a noninfective meal 2-4 days later. The mosquitoes were dissected 9-10 days after they received the infective blood meal, and the sporontocidal effect of the drugs was evaluated by the number of oocysts found in the gut. Both cycloguanil and pyrimethamine had marked sporontocidal activity. The susceptibility pattern of the strains to the sporontocidal effect of pyrimethamine and cycloguanil was similar to the susceptibility of their asexual blood stages in vitro to the schizontocidal effect of the compounds. The sporontocidal effect was observed only when the compounds were administered at the same time as the infective blood meal, but not when they were given 2-4 days later. No sporontocidal activity was observed with primaquine. This system permits more reliable quantitative observations than have been possible with previous methods.

In vivo efficacy of chloroquine treatment for Plasmodium falciparum in Malawian children under five years of age.

In 1984 the government of Malawi instituted a program to reduce malaria mortality and morbidity in children less than 5 years of age as a part of the Combatting Childhood Communicable Diseases (CCCD) program. To define the appropriate malaria therapy regimen, investigators used a quality assurance design in a simplified 7-day in vivo drug response study with follow-up observations on day 2 (D2), D3, and D7 after the initial day of the study (D0). The efficacy of oral chloroquine was assessed in 224 children who were enrolled at 6 sites, 2 in each of the 3 administrative regions of Malawi. Parasitological failure, defined as failure of parasitemia to decrease by 75% of the value by D3 or presence of any detectable parasitemia on D7, ranged from 41%-65% following administration of chloroquine 25 mg (base)/kg. However, only 8% of children who were parasitemic on D7 were febrile or judged to be ill. Considering these therapeutic results and the higher cost and limited availability of alternative therapies, chloroquine 25 mg/kg therapy was adopted as the primary therapy for malaria.