Chloroquine resistance produced in vitro in an African strain of human malaria.

After condinous cultivation in the presence of chloroquine, an African strain of the malaria parasite, Plasmodium falciparu, acquired resistance to the drug. The resistance was stable and comparable in vitro to that occurring naturally in a strain from Southeast Asia. This suggests that chloroquine resistance, absent until now in Africa, might arise in the future.

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.

Acute HIV illness following blood transfusion in three African children.

Three children are described in whom pre-transfusion samples were HIV-seronegative and post-transfusional samples, obtained within 1 week after transfusion, were HIV-seropositive. Two of them developed a transient fever within 1 week of receiving the blood transfusion, and a transient generalized skin eruption which lasted for about 2 weeks. All three developed persistent generalized lymphadenopathy. One child developed a lumbar herpes zoster 7 months after transfusion. IgM Western blots demonstrated the presence of antibodies to protein bands p17, p24 and p55 in all three children. These three case reports suggest that children who receive a seropositive blood transfusion are at high risk for developing acute manifestations of HIV infection.

Trends and risk factors for HIV-1 seropositivity among outpatient children, Kinshasa, Zaire.

To investigate recent trends in pediatric HIV-1 infection and the early impact of a blood screening program begun in one hospital in 1987 in Kinshasa, Zaire, we evaluated 1110 consecutive children seen in the pediatric emergency ward of the city's largest hospital in November 1988. The HIV-1 seroprevalence was 5.0%, not significantly higher than the rate of 3.8% found in 1986 (P = 0.2). The seropositivity rate was bimodally distributed; children less than 6 months of age had a higher rate (12.6%) than children 6-11 months old (1.9%; OR = 7.6; P less than 0.0001) and children 1-13 years old (4.1%; OR = 3.4; P less than 0.0001). Seropositive children greater than or equal to 1 year of age were more likely than seronegative children to be anemic and to have signs of malnutrition. A previous blood transfusion was associated with HIV-1 seropositivity among children greater than or equal to 1 year of age (OR = 5.4, P less than 0.0005), but not among younger children. Fifty-two per cent of seropositive children greater than or equal to 1 year of age received a transfusion (etiological fraction = 42%). The association with seropositivity was higher for those who had received a transfusion before 1987 than for those who had received a transfusion since 1987 (OR = 4.8, P = 0.01). These findings suggest a relatively stable, high pediatric HIV-1 seroprevalence in Kinshasa and a decreased but continued risk of transfusions. Expansion of currently limited blood transfusion screening programs, and the development of new strategies for limiting transfusions and preventing severe anemia, are needed.

PIP: To investigate recent trends in pediatric HIV-1 infection and the early impact of a blood screening program begun in 1 hospital in Kinshasa, Zaire, the authors evaluated 1110 consecutive children seen in the pediatric emergency ward of the city's largest hospital in November 1988. The HIV-1 seroprevalence was 5.0%, not significantly higher than the 3.8% rate found in 1986 (p=0.2). The seropositivity rate was bimodally distributed; children 6 months of age had a higher rate (12.6%) than children 6-11 months old (1.9%; OR+7.6; p0.0001) and children 1-13 years old (4.1%; OR+3.4; p0.0001). Seropositive children or= 1 year of age were more likely than seronegative children to be anemic and to have signs of malnutrition. A previous blood transfusion was associated with HIV-1 seropositivity among children or= 1 year of age (OR=5.4, p0.0005), but not among younger children. 52% of seropositive children or= 1 year of age had received a transfusion (etiological fraction=42%). The association with seropositivity was higher for those who had received a transfusion before 1987 than for those who received 1 since that time (OR=4.8, p=0.01). These findings suggest a relatively stable, high pediatric HIV-1 seroprevalence in Kinshasa and a decreased but continuous risk of transfusions. Expansion of currently limited blood transfusion screening programs and the development of new strategies for limiting transfusions and anemia prevention are necessary.

Evaluation of a clinical case definition of AIDS in African children.

In July 1986, a provisional clinical case definition of AIDS in children, developed by the World Health Organization (WHO) for surveillance purposes in Africa, was tested on 159 patients hospitalized in the Department of Pediatrics at Mama Yemo Hospital, Kinshasa, Zaire. Twenty-one (13%) of these children were seropositive for HIV. In this population, the clinical case definition of pediatric AIDS was found to be fairly specific (87%) but lacked sensitivity (35%). The positive predictive value for HIV seropositivity was 25%. This study suggests that it is more difficult to define AIDS clinically in children than in adults and that the utility of the proposed WHO clinical case definition for pediatric AIDS for surveillance of children's AIDS in Africa is limited.

Plasmodium falciparum in vitro: determination of chloroquine sensitivity of three new strains by a modified 48-hour test.

Three recently isolated African strains of Plasmodium falciparum were tested in vitro for their response to chloroquine. Both the 48-hour method described earlier and a modified 48-hour test were used, yielding comparable results. Strain FCR-7/Kenya, isolated from a clinically chloroquine-resistant case, was more resistant to the drug in vitro than the two other strains (FCR-8/West Africa and FCN-1/Nigeria, both isolated from chloroquine-sensitive cases). Complete inhibition of parasite growth occurred for strain FCR-7/Kenya in a drug concentration range ten times higher than for strains FCR-8/West Africa and FCN-1/Nigeria. In the modified 48-hour test, a lower erythrocyte suspension (2%) allows continuous growth of the parasites over a 48-hour cycle without necessitating change in medium. It thus offers distinct advantages for routine laboratory work as well as for potential field trials.

A simple field kit for the determination of drug susceptibility in Plasmodium falciparum.

A field kit has been developed which greatly simplifies the performance of the 48-hour in vitro test for drug resistance in Plasmodium falciparum. The kit uses an easily reconstituted lyophilized culture medium, and requires only a fingerprick blood sample. In parallel tests with 13 isolates of P. falciparum in Haiti, the new technique had a success rate equal to that of the previously described method, with comparable results in terms of parasite susceptibility in vitro to chloroquine and pyrimethamine.

In vitro cultivation of exoerythrocytic stages of the human malaria parasite Plasmodium malariae.

Exoerythrocytic stage parasites of Plasmodium malariae were obtained in vitro by inoculating primary cultures of hepatocytes from a chimpanzee (Pan troglodytes) and a monkey (Aotus lemurinus griseimembra) with sporozoites. Schizonts were observed in chimpanzee hepatocytes 8, 11, and 13 days after inoculation. Only 1 schizont was seen in Aotus hepatocytes at day 13. The morphology and development rates of P. malariae exoerythrocytic stages obtained in vitro were similar to those previously described in vivo.

In vitro activity of antimalarial compounds on the exoerythrocytic stages of Plasmodium cynomolgi and P. knowlesi.

Primary cultures of Macaca mulatta hepatocytes infected with sporozoites of Plasmodium knowlesi, P. cynomolgi (Cambodian strain), and P. cynomolgi bastianellii were exposed in vitro to 7 antimalarial compounds. The number of exoerythrocytic schizonts present after 4-7 days of culture was used to assess the activity. With pyrimethamine, proguanil, cycloguanil, primaquine, and 2 of its analogues (WR242511 and WR238605), marked inhibition of schizont formation could be achieved at concentrations below those causing a cytotoxic effect on the host hepatocytes. Chloroquine had only minimal schizonticidal activity at a concentration that produced severe hepatocyte toxicity. This simian in vitro system provides a reliable model for screening antimalarial compounds and for investigating their effects on the hepatic stage of malaria parasites.

Plasmodium falciparum in Kinshasa, Zaire: in vitro drug susceptibility studies.

In June 1986, Plasmodium falciparum parasites were collected from 33 children presenting at the Mama Yemo Hospital in Kinshasa (Zaire) and were successfully tested in vitro by a 48-hr reinvasion test for their susceptibility to various antimalarial drugs. In vitro resistance to chloroquine was found in 82% of the isolates, a marked increase over findings obtained by the same technique 3 years ago in Kinshasa. In vitro chloroquine resistance was not associated with a history of previous drug intake. The inhibitory endpoints for quinine varied from 0.03 to 1 microM, and correlated with the chloroquine endpoints in the corresponding isolates (r = 0.64). Pyrimethamine resistance in vitro was demonstrated in 52% of the isolates tested.

Antibodies to Pf155, a major antigen of Plasmodium falciparum: longitudinal studies in humans.

Antibodies to Pf155, a major Plasmodium falciparum antigen detected in the membrane of glutaraldehyde-fixed and air-dried erythrocytes infected with P. falciparum, were studied in serum samples collected from patients treated for neurosyphilis by induced P. falciparum infection. In 3 patients with no previous documented exposure to malaria, the antibodies were detected late and reached low titers. In 5 patients with extensive previous malaria infections, the antibodies appeared rapidly and reached high titers. The immunofluorescence findings were confirmed by immunoblots. No correlation was observed between antibodies to Pf155 and antibodies detected by standard immunofluorescence with whole parasite antigen.

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.

Cultivation of exoerythrocytic stages of Plasmodium cynomolgi, P. knowlesi, P. coatneyi, and P. inui in Macaca mulatta hepatocytes.

Exoerythrocytic stage parasites of Plasmodium cynomolgi, P. knowlesi, P. coatneyi, and P. inui were cultured by inoculating primary cultures of hepatocytes from Macaca mulatta with sporozoites. Less than 1% of inoculated sporozoites survived. Morphology and size of the liver stages in all 4 species were similar to in vivo descriptions and the time required for in vitro maturation correlated well with prepatent periods described.

Field evaluation in Kenya of a 48-hour in vitro test for Plasmodium falciparum sensitivity to chloroquine.

A 48-hour in vitro test for determining the chloroquine sensitivity of Plasmodium falciparum isolates was evaluated in Kisumu and Malindi, Kenya. P. falciparum isolates from 14 children, aged 5 to 13 years, were studied. In vivo and 48-hour in vitro tests were done on all 14. Successful Rieckmann macro and micro in vitro tests for chloroquine sensitivity were completed in nine isolates each. All 14 infections cleared within 3 days of beginning chloroquine treatment, and none recrudesced during a 7-day (8 patients) or 28-day (6 patients) follow-up period. The three in vitro tests gave comparable results. Although all isolates tested were chloroquine sensitive in vitro, different response patterns were observed. In the 48-hour test, 10 isolates were inhibited at chloroquine concentration less than or equal to 0.03 nmol/ml medium. These isolates were inhibited by less than or equal to 0.5 nmol of chloroquine per ml blood in the Rieckmann macro test and by 2-6 pmol/well in the micro test. The other four isolates had response patterns intermediate between those of previously reported sensitive and resistant strains. Complete inhibition did not occur until chloroquine concentrations of greater than or equal to 0.03 nmol/ml medium in the 48-hour test, greater than or equal to 0.5 nmol/ml blood in the macro test, and 6 pmol/well in the micro test. The results demonstrate that the 48-hour test is a useful addition to existing in vivo and in vitro methods for determining the chloroquine sensitivity of P. falciparum in the field.

Monitoring of chloroquine sensitivity of Plasmodium falciparum in Haiti, 1981-1983.

Between 1981 and 1983, in vivo and in vitro studies were conducted in Haiti to assess the responsiveness of Plasmodium falciparum to chloroquine. The standard tests successfully performed included 92 WHO standardized in vivo field tests and 160 in vitro tests (64 macrotests, 33 microtests, and 63 48-hr tests). No clearcut evidence of chloroquine resistance was detected. In 3 in vivo and 5 in vitro tests, a decreased susceptibility to the drug was suggested, but these isolated findings failed to be corroborated by parallel alternate tests. In addition, during the initial trial of an alternate monitoring system, 339 simplified 7-day in vivo tests were successfully performed, with no suggestion of resistance detected. This simplified 7-day in vivo test potentially represents an efficient low cost method for monitoring drug resistance in many developing countries.

Intravenous quinine therapy of hospitalized children with Plasmodium falciparum malaria in Kinshasa, Zaire.

To examine the clinical and parasitologic efficacy of quinine, we studied 34 children (7 months-13 years old) with severe or moderately severe Plasmodium falciparum infections. Quinine 10 mg/kg every 8 hr for 3 days was administered, initially by intravenous infusion of quinine formate followed by oral quinine dihydrochloride when tolerated. Thirty-three of the 34 patients were clinically well and had negative malaria smears 7 days after the initiation of therapy; 1 child, who presented in coma, died 29 hr after enrollment. The mean fever clearance time was 44.1 hr, and the mean parasite clearance time was 59.6 hr. A mean peak quinine level of 9.7 ppm was attained after the second dose of quinine, and the minimum concentration was maintained at 5-7 ppm during the 2nd and 3rd hospital days. In vitro testing was conducted with parasites from 10 patients: 9 isolates were resistant to chloroquine, and inhibition of schizont development with quinine occurred at a concentration of 8-32 pmol/well.

Plasmodium falciparum-associated anemia in children at a large urban hospital in Zaire.

Chloroquine-resistant Plasmodium falciparum malaria and human virus (HIV) infection through blood transfusions used to treat malaria-associated anemia are causes of increasing morbidity and mortality among children in Africa. To evaluate the role of malaria and other risk factors for pediatric anemia, we conducted a study of children brought to the emergency ward of a large urban hospital in Kinshasa, Zaire. A total of 748 children ages six through 59 months were enrolled; 318 (43%) children were anemic (hematocrit < 33%), including 74 (10%) who were severely anemic (hematocrit < 20%). Plasmodium falciparum parasites were detected in 166 children (22%); hematocrits for these children (mean 25.8%) were significantly lower than for aparasitemic children (mean 33.7%; P < 10(-6)). Fever with splenomegaly (odds ratio [OR] = 6.5, P = 0.02), parasitemia (OR = 3.5, P < 0.001), lower socioeconomic status (OR = 2.0, P = 0.004), and malnutrition (OR = 1.8, P = 0.06) were independently associated with anemia in a multivariate model. Recent antimalarial therapy was also associated with a lower hematocrit, suggesting that chloroquine may have aggravated the anemia. A reassessment of the effectiveness of strategies to diagnose and treat malaria and malnutrition is necessary to decrease the high prevalence of anemia and the resultant high rate of blood transfusions in areas endemic for malaria and HIV.

Malaria, intestinal parasites, and schistosomiasis among Barawan Somali refugees resettling to the United States: a strategy to reduce morbidity and decrease the risk of imported infections.

In 1997, enhanced health assessments were performed for 390 (10%) of approximately 4,000 Barawan refugees resettling to the United States. Of the refugees who received enhanced assessments, 26 (7%) had malaria parasitemia and 128 (38%) had intestinal parasites, while only 2 (2%) had Schistosoma haematobium eggs in the urine. Mass therapy for malaria (a single oral dose of 25 mg/kg of sulfadoxine-pyrimethamine) was given to all Barawan refugees 1-2 days before resettlement. Refugees >2 years of age and nonpregnant women received a single oral dose of 600 mg albendazole for intestinal parasite therapy. If mass therapy had not been provided, upon arrival in the United States an estimated 280 (7%) refugees would have had malaria infections and 1,500 (38%) would have had intestinal parasites. We conclude that enhanced health assessments provided rapid on-site assessment of parasite prevalence and helped decrease morbidity among Barawan refugees, as well as, the risk of imported infections.