Complete development of hepatic stages of Plasmodium falciparum in vitro.

An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

Patterns of in vitro resistance to chloroquine, quinine, and mefloquine of Plasmodium falciparum in Cameroon, 1985-1986.

The drug sensitivity of 246 Plasmodium falciparum isolates was studied in vitro in five areas of Cameroon at the end of 1985. Results demonstrate that parasites resistant either to chloroquine, quinine, or mefloquine, or to two of these drugs, were prevalent in four of the areas investigated, but the drug response pattern varies widely from one area to another. The recent explosive emergence of chloroquine resistance in the south of the country, where both prevalences and levels are very high (up to 86%), contrasts with only moderate levels of resistance in the north. This may be related to differences in transmission by mosquitoes between Sahel and forest areas. Quinine resistance was observed in 24% of the isolates studied in vitro and was frequently associated with chloroquine resistance. The presence of isolates responding poorly to mefloquine, observed mainly in northern Cameroon, suggests that resistance may occur in areas where the drug has never been used.

Decreased sensitivity to chloroquine and quinine of some Plasmodium falciparum strains from Senegal in September 1984.

The in vitro sensitivity of 135 Plasmodium falciparum isolates collected in the areas of Thies and Kaolack, Senegal, were studied in September 1984, by means of the 48 hr microtest with 3H-hypoxanthine incorporation. Results are available in 110 of 135 cases studied (81%). The isolates collected around Kaolack were found on average less sensitive to chloroquine than those from Thies (mean EC50 28 and 17 nmol/l of medium, respectively, P less than 0.05) and in 1 isolate a high degree of resistance was found (EC50 178 nmol/l). Some of those isolates also were studied using the WHO standard microtest and similar results were recorded. With both assays probit regression lines show EC99 in Kaolack greater than 114 nmol/l. These data suggest that in 1984 chloroquine resistance was possibly emerging in the extreme west of Africa. Sensitivity to quinine could be evaluated in 15 of 24 isolates tested. One of them, originating from Thies, was highly resistant to this drug (EC50 760 nmol/l) and 2 additional isolates with EC50s of 370 and 274 nmol have decreased sensitivity. These results suggest that quinine, as well as chloroquine, sensitivity should be monitored in the African region.

High level of sensitivity to chloroquine of 72 Plasmodium falciparum isolates from southern Cameroon in January 1985.

The sensitivity to chloroquine, quinine and mefloquine of Plasmodium falciparum isolates from 3 areas of southwest Cameroon was evaluated using an in vitro microtest with estimation of parasite growth by 3H-hypoxanthine incorporation. Among 2,429 children examined, P. falciparum was found on thin smears in 124 of them, 76 isolates were submitted to in vitro tests and 72 were successful. In the locations studied, some of which are close to the area of Limbe where in vivo resistance has been reported, all 72 isolates were found fully sensitive to low concentrations of chloroquine (mean EC50 5.9 ng/ml or 18.5 nmol/l of medium). In 47 of these isolates simultaneously tested using WHO microtest predosed plates, the sensitivity was identical. Out of 39 tests performed with quinine, 35 were successful. While most strains responded to low concentrations, some showed a decreased sensitivity to the drug, the EC50 of 4 of them being in the range 230-300 nmol/l. Each of the 17 isolates tested with mefloquine was susceptible to very low concentrations of freshly prepared drug solution. While chloroquine-resistant strains may already exist in Cameroon, the present study suggests that they would be restricted to a limited area and are not widespread. Data also suggest that monitoring of the sensitivity of P. falciparum to quinine might soon be necessary.

[Plasmodium falciparum drug resistance in the Congo. Evaluation of surveys carried out from 1985 to 1989]. / La résistance médicamenteuse de Plasmodium falciparum au Congo. Bilan des enquêtes réalisées de 1985 à 1989.

Surveys on drug sensitivity of Plasmodium falciparum carried out between 1985 and 1989 included 7-day in vitro tests and in vivo tests. 485 in vivo tests were carried out in eight surveys conducted in Brazzaville and in several inland regions. The subjects were congolese children aged between 3 months and 15 years old. They were recruited in hospital, mother-child clinics or at school. The drugs studied were chloroquine, amodiaquine and the sulfadoxine-pyrimethamine combination. 182 strains were tested in vitro in two surveys (December 1985 and January 1987); amino-4-quinolines, quinine and mefloquine were studied. Although resistance to amino-4-quinolines is a recent occurrence, by 1985 it had spread widely in the indigenous population in the Centre and South of the country. Resistance has since increased gradually, especially for chloroquine which undergoes specific surveillance. The situation is less serious in the North, a less densely populated region which is still enclosed. In an in vivo comparative study with chloroquine conducted in Brazzaville in November 1986, amodiaquine was found to be only slightly more effective at a similar dosage. At that time, certain isolated observations already seem to imply that the sulfadoxine-pyrimethamine combination was also affected by resistance. This was not corroborated in an in vivo study carried out in 1989 on 40 children presenting with a malarial attack. Although the sensitivity to quinine may probably be decreased. This drug cannot yet be considered as being truly affected by resistance. The activity of mefloquine, the use of which is still limited, was satisfactory in 1987 in two different regions of the country.

[Plasmodium falciparum and drepanocytic gene in Popular Republic of Congo. I. Prevalence of malaria and drepanocytic trait among school children in Brazzaville area (author's transl)]. / Le paludisme à Plasmodium falciparum et le gène de la drépanocytose en République Populaire du Congo. I. -- Prevalence du paludisme et du trait drepanocytaire, en milieu scolaire dans la région Brazzavilloise.

From 1978 to 1979, 5 surveys, among schoolchildren, were carried out during the rainy season in the neighbourhood of Brazzaville (R.P. Congo): 3 in PK 45 village (northern part of the capital), 2 in Djoumouna village (southern part), and 1 in "Talangai" (a suburb of the capital). 868 exams (plasmodic and splenic index fitted with hemoglobin composition [Hb AA or Hb AS]) were done. It appeared that 19,6% of schoolchildren examined were heterozygous sicklers (AS). This percentage confirmed the previous results from other authors in different countries of Central Africa. On the other hand, in spite of an intense transmission, both plasmodic and splenic index were, on the average, relatively low (24,5 and 24,8% respectively). Plasmodium falciparum was largely predominant (95,3% of infections) but P. ovale and P. malariae were also found (1,9% for each species). From our study no obvious "protecting effect" can be attributed to sickle cell trait because plasmodic index of children AA and AS were similar (23,8 and 27,6% respectively). A slight decrease of splenic index was noticed in AS in regard to AA (19,4 and 26.1% respectively). It is difficult to consider this no significative regression as a definitive proof of a premunition stronger in AS than in AA. Effectively some splenic infarctus are well known to be a regular physiopathological process occurring in homozygous SS but often in heterozygous AS too. In such highly endemic and stable malaria area the problem of a suitable antimalaria strategy remains to be solved.

Activity of a combination of three cinchona bark alkaloids against Plasmodium falciparum in vitro.

In vitro studies with quinine, quinidine, cinchonine, and cinchonidine showed that despite a similarity of chemical structure, the effectiveness of these cinchona bark alkaloids against several culture lines of Plasmodium falciparum varied widely. Depending on the strain tested, quinidine and cinchonine were 1 to 10 and 1 to 5 times, respectively, more active than quinine. A combination made of equal parts of quinine, quinidine, and cinchonine was found to have several interesting features; it had activity similar to that of quinine against quinine-susceptible strains but was found to be 2 to 10 times more effective against strains resistant to quinine and had a more consistent effect than any of the alkaloids used singly. The potentiation was found to depend mainly on the presence of cinchonine in the mixtures studied. Synergism was also confirmed in a study of 25 P. falciparum strains isolated from Thai patients. Combinations of cinchona bark alkaloids could thus be of interest in areas where P. falciparum is becoming less susceptible to quinine.

One-step Plasmodium falciparum cultivation--application to in-vitro drug testing.

P. falciparum was cultivated by a one-step method which requires medium renewal and addition of fresh RBC only every three days and allows high proliferation rates of the parasite. The method has been used for routine maintenance of strains and applied to the in-vitro evaluation of the effect of antimalarial compounds over 3 days periods as measured by 3H-Hypoxanthine incorporation.

Widespread in vitro resistance to chloroquine of Plasmodium falciparum in the Congo, 1987.

The drug sensitivity of 184 Plasmodium falciparum isolates was studied in vitro in three areas of the Congo in January 1987. Results show that parasites resistant to chloroquine but not to quinine or mefloquine were prevalent in the three investigated regions, but the drug response pattern varied widely. In Brazzaville, after the outburst of chloroquine resistance in 1985, prevalence of chloroquine resistant isolates seemed to have stabilized around 60%. The phenomenon more recently reached the North where about 30% isolates could be considered as drug resistant. As in Cameroon, wide variations in the prevalence and the level of resistance were observed within a very limited area emphasizing the role of drug pressure in market places where chloroquine is easily available.

[Is it necessary to perform sensitivity tests for malaria in the hospital environment?]. / Faut-il pratiquer un antipaludogramme en milieu hospitalier?

From January 1984 to June 1985, 20 out of 65 P. falciparum strains isolated in an hospital in Paris were epidemiologically suspect of resistance to chloroquine. 15 of them were submitted to in vitro chemosensitivity tests with several antimalarials drugs. In cases of suspected chloroquine resistance the initial choice of an alternative drug is mainly presumptive and is generally not guided by the in vitro chemosensitivity assay which results are too much delayed. When in vitro assay shows sensitivity to amino-4-quinolines treatment can be modified accordingly. The determination of plasma concentration of drugs are required in any case. However in vitro sensitivity assays would probably appears essential to physicians in cases of resistance to quinine.

Efficacy of intramuscular amopyroquin for treatment of Plasmodium falciparum malaria in the Gabon Republic.

The efficacy of a 12-mg/kg (of body weight) intramuscular amopyroquin (ApQ) regimen (two successive 6-mg/kg injections at a 24-h interval), previously established from kinetic studies on healthy volunteers and multicenter studies on patients with malaria, was investigated in 152 patients (children and adults) in Gabon with Plasmodium falciparum malaria. All children in the present study (ages, 1 to 14 years) showed higher degrees of parasitemia and temperatures and lower hematocrit values than did adults at the time of admission. No major side effects in the patients were observed. On day 7, all patients were apyretic; clearance of parasites was obtained in 143 of 152 patients (94%); a low level of parasitemia was observed in nine patients, all of whom were children (6%). In vitro chemosusceptibility tests carried out on P. falciparum isolates from patients demonstrated 51% of resistance to chloroquine (Cq). A correlation was found between the in vitro chemosusceptibilities to Cq and ApQ, but no relationship between the in vitro activity and the in vivo efficacy of ApQ could be found. Concentrations of ApQ in blood assayed by high-performance liquid chromatography on day 2 did not differ significantly between the groups in whom therapy was a success or a failure, although the mean ApQ concentration in blood for the group that failed therapy was 31% lower. Concentrations greater than 100 nmol of self-prescribed Cq and amodiaquine per liter, which were assayed simultaneously with ApQ, were observed in 78 patients (51%). They did not correlate with degrees of parasitemia compared with ApQ alone, which did. Successful treatment by day 7 was obtained in 69 of 74 patients (93%) who had no other 4-aminoquinolines in their blood. The results of the present study show that an ApQ regimen of 12 mg/kg over 2 days may be an alternative for the treatment of Cq-resistant malaria, at least in adult patients, in the field.

[Development of drug resistance in cases of P. falciparum malaria of African origin in a Parisian hospital. Comparison with field data and therapeutic consequences]. / Evolution de la chimiorésistance des cas de paludisme a P. falciparum d'origine africaine dans un hôpital parisien. Comparaison avec les données observées sur le terrain et conséquences thérapeutiques.

The number of P. falciparum malaria cases, contracted in Africa, diagnosed by the department of Parasitology and Tropical diseases of the Pitié-Salpêtrière Hospital Group has shown a significant increase in 1985-1986 when compared with the 15 past years (+23%). This fact is related to the spread of chloroquine or amodiaquine resistant falciparum malaria from East Africa (1980-1983: 54%) to Central Africa (1985: 54.5%) and now to a country of west Africa (Benin 1986).

[Culture systems for production of promastigote and amastigote forms of Leishmania. Application to serological diagnosis and therapeutic trials]. / Etude de systèmes de culture pour la production des formes promastigotes et amastigotes des Leishmanies. Application au diagnostic sérologique et aux essais thérapeutiques.

Several species of leishmania and three methods of cultivation: monophasic, biphasic and co-cultivation were used in a compared study bearing on the intensive production of leishmania. In addition by applying, a new in vivo model, comprising an injection of sarcomatous cells and promastigotes into BALB/c mice and also an extraction on a discontinuous gradient (Radioselectan 60%), it was possible to obtain highly purified isolates of amastigote forms. The use of two antigens: promastigotes and amastigotes, is to be recommended for the serological diagnosis, by indirect immunofluorescence, of kala-azar. The new in vivo model merits further consideration for research concerning new molecules active against leishmania.

[Obtaining hepatic stages of Plasmodium falciparum in vitro]. / Obtention in vitro des stades hépatiques de Plasmodium falciparum.

Sporozoites of Plasmodium falciparum, obtained by membrane feeding of Anopheles freeborni or A. stephensi with cultured gametocytes, were used to infect monolayers of human hepatocytes. Fluorescent labelling with an African serum as well as Giemsa staining performed from day one to day 7 of cultures, demonstrated the presence of numerous hepatic schizonts measuring up to 40 micron.