Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria: II. Correlation of in vitrowith in vivo sensitivity

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0·5 to 0·8 nmol/ml to inhibit completely maturation from ring forms to schizonts.This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria

Thirty-five children with Plasmodium falciparum malaria were treated with 25 mg/kg chloroquine over three days and observed for seven days during which blood films were examined daily for malaria parasites.Asexual forms of P. falciparum which were present in the blood films of all the patients before commencing treatment disappeared rapidly from the blood so that by the third day no parasites were seen in the blood film. The blood films remained negative for the rest of the seven-day observation period.Plasma chloroquine determination in eight of the patients showed high blood levels during the first three days.The results do not confirm the suspicion of chloroquine-resistant P. falciparum in the area studied although RI level of resistance by WHO criteria was not excluded.     

Fansidar resistant falciparum malaria acquired in South East Asia

A case of Plasmodium falciparum malaria resistant to Fansidar (sulphadoxine plus pyrimethamine) at a level corresponding to R III and resistant to chloroquine is reported. The infection was most certainly acquired in Malaysia, but diagnosed and treated in a non-malarious area.Normal resorption and elimination rates of the Fansidar components excludes cure failure due to abnormal drug fate in the host.P. falciparum parasites from the patient have been maintained in in vitro cultures.The patient was permanently cured with mefloquine.     

Serum concentrations of chloroquine in a patient with a late recrudescence of Kenyan Plasmodium falciparum malaria

A Swedish tourist who had visited Kenya fell ill with Plasmodium falciparum malaria 11 days after returning home, in spite of taking pyrimethamine (50 mg weekly) as malaria prophylaxis. Chloroquine treatment (25 mg base/kg body-weight) giving serum concentrations of 0·30 μmol/l cleared the patent parasitaemia and the patient recovered. Recrudescence occurred, however, within 42 days. A second chloroquine course (30 mg base/kg) gave serum levels up to 1·28 μmol/1. The patient improved rapidly and remained healthy during 28 days without renewed parasitaemia. Further follow-up for 10 months was uneventful. We consider it urgent to assess chloroquine concentrations in serum in patients being treated for falciparum malaria in order to obtain data on fully effective levels. Ineffective serum levels should be ruled out in cases not responding to chloroquine, especially when chloroquine-resistance is suspected.     

Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti

Plasmodium falciparum parasites have been endemic to Haiti for >40 years without evidence of chloroquine (CQ) resistance. In 2006 and 2007, we obtained blood smears for rapid diagnostic tests (RDTs) and filter paper blots of blood from 821 persons by passive and active case detection. P. falciparum infections diagnosed for 79 persons by blood smear or RDT were confirmed by PCR for the small subunit rRNA gene of P. falciparum. Amplification of the P. falciparum CQ resistance transporter (pfcrt) gene yielded 10 samples with amplicons resistant to cleavage by ApoI. A total of 5 of 9 samples had threonine at position 76 of pfcrt, which is consistent with CQ resistance (haplotypes at positions 72–76 were CVIET [n = 4] and CVMNT [n = 1]); 4 had only the wild-type haplotype associated with CQ susceptibility (CVMNK). These results indicate that CQ-resistant haplotype P. falciparum malaria parasites are present in Haiti.     

Sensitivity of Plasmodium falciparum to chloroquine in Busia District,Kenya

Individuals infected with Plasmodium falciparum were randomly divided into two groups; one group was treated with a single dose of 10 mg chloroquine base per kg. body-weight and the other with 25 mg base of chloroquine per kg body-weight given over three days, followed by an observation period of seven days. By Day 3 of observation complete parasite clearance had occurred in all the 125 triple dose recipients and 113 of 114 (99·1%) of those who had the single dose.94·4% of 36 isolates tested in vitro by the macrotechnique were sensitive to drug concentration of 0.75 nmol/ml blood or less. One isolate was relatively less sensitive and required a concentration of chloroquine of 1·50 nmol/ml to inhibit schizont growth. However, the same isolate responded well to 25 mg base of chloroquine. These findings have demonstrated that, at present, isolates of P. falciparum in Busia District are sensitive to a standard dose of 10 mg chloroquine base and there is no reason therefore to resort to alternative antimalaria drugs. These should be reserved for special cases only.     

Chloroquine tolerance of Ethiopian strains of P. falciparum

11 of 41 Ethiopians in hospital with P. falciparum malaria experienced a delayed recrudescence of parasitaemia following treatment with 10 mg. chloroquine base per kg. of body weight. Parasites from 25 of the 41 patients were successfully cultivated in vitro, and 9 isolates showed development in chloroquine concentrations of 0·5 to 1·0 millimicromoles per c.c. of blood. 3 isolates with development at the 1·0 millimicromole level were from patients who experienced a recrudescence of parasitaemia. In vivo and in vitro results suggest a chloroquine responsiveness of some Ethiopian isolates of P. falciparum which is between that of the sensitive Uganda I strain and the resistant Malayan (Camp.) strain; a finding not previously documented in African parasites.     

Plasmodium falciparum sensitivity to chloroquine in the Buyo Region, Ivory Coast

As part of a project, supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, for achieving a reduction of mortality due to Plasmodium falciparum by means of a primary health care programme, in vivo and in vitro sensitivity testing of P. falciparum to chloroquine was carried out in the Buyo Region of the Ivory Coast. Blood samples from a total of 595 children aged 2-13 years from 5 villages were screened microscopically and 32 of these children were selected for in vivo testing and 36 for in vitro testing. All 32 in vivo test patients were treated with 25 mg chloroquine base per kg, given in divided doses over 3 days, and all of them completed the 7-day observation period. Daily blood slides were taken and these were negative for asexual parasites on days 3 to 7. The mean parasite clearance time was 1.8 days. The 36 in vitro tests produced satisfactory results in 19 isolates. Complete inhibition was achieved in all 19 at 5.7 pmol chloroquine base per well (1.14 μmol/l of blood); 7 of these isolates showed complete inhibition at 1 pmol, 15 at 2 pmol and 17 at 4 pmol.     

Prospective double-blind trial of two different doses of mefloquine plus pyrimethamine-sulfadoxine compared with pyrimethamine-sulfadoxine alone in the treatment of falciparum malaria

This double-blind study is based on the treatment of 75 adult male patients suffering from Plasmodium falciparum malaria in Medellín, Colombia, a city in which there is no malaria transmission. The patients, who came from regions with high resistance to antimalarials, were divided into three groups receiving single-dose treatment as follows: a combination of 280 mg mefloquine, 800 mg sulfadoxine and 40 mg pyrimethamine; a combination of 420 mg mefloquine, 1200 mg sulfadoxine and 60 mg pyrimethamine; and a combination of 1500 mg sulfadoxine and 75 mg pyrimethamine. After treatment, follow-up examination was performed daily for I week and then weekly for another 3 weeks. The cure rate in the mefloquine groups (within the study period of 28 days) was 100%, and in the third group 75%. Normal blood levels of the administered drugs were found in 6 patients of the third group who were not cured; they were subsequently cured with a single dose of 1000 mg of mefloquine. Drug tolerance was good and no toxic effects were demonstrated in blood and urine examinations. While the doses in the drug combinations (containing mefloquine) gave very good results, we would recommend a slightly higher dose combination of mefloquine with sulfadoxine—pyrimethamine for the treatment of falciparum malaria in areas with a high prevalence of chloroquine resistance.     

Chloroquine resistant Plasmodium falciparum in indigenous residents of Cameroon

Thirty-nine percent (36 of 92) of children in Limbe, Cameroon, treated with chloroquine (10 mg/kg body weight on days 1 and 2, and 5 mg/kg on day 3) for falciparum malaria failed to respond within 7 d of treatment. Twenty-two of these children with chloroquine-resistant malaria were successfully treated with Fansidar [one-half tablet (250 mg sulfadoxine and 25 mg pyrimethamine) per 10 kg body weight], while the other 14 children were cured with mefloquine (25 mg/kg body weight). In vitro, a combination of verapamil at 1.0 x 10(-6) M with chloroquine or desethylchloroquine reversed resistance to the antimalarial drug and its primary metabolite in each of the 2 isolates successfully adapted and maintained in continuous culture. Similar combinations had no effect on susceptibilities of a sensitive reference clone, D6, used as control. Both chloroquine-resistant isolates from Cameroon were significantly more susceptible to mefloquine and halofantrine in vitro than the chloroquine-sensitive reference clone. Clinical observation, and in vitro confirmation, of chloroquine-resistant falciparum malaria in these indigenous children from Cameroon, and the current socio-economic condition in West Africa, underscore the need for pragmatic health management policies for efficient use of alternative antimalarial drugs in controlling drug resistant Plasmodium falciparum in the region. This observation of reversal of chloroquine resistance in isolates of P. falciparum obtained from West Africa, and a previous report on clones obtained from south-east Asia and South America, suggest that the mechanism(s) of resistance to chloroquine may be identical in resistant parasites from the 3 continents.     

In vivo response of Plasmodium falciparum to chloroquine in pregnant and non-pregnant women in Siaya District, Kenya

Chemoprophylaxis using chloroquine (CQ) in suppressive doses has been recommended to protect pregnant women in malarious areas from the adverse effects of malaria during pregnancy. In a malaria-endemic area of western Kenya with CQ-resistant Plasmodium falciparum, we determined the prevalence and density of falciparum infection in gravid and nulligravid women and compared the in-vivo parasite response to CQ using two regimens: 25 mg/kg body weight (CQ25) divided over a period of three days (for high-density parasitaemias) and 5 mg/kg body weight (CQ5) weekly for 4 weeks (for low-density parasitaemias). P. falciparum infections were present in 102 (42%) of 244 pregnant women. A greater proportion of primigravidae were parasitaemic (68%) than nulligravidae (50%, P=0.02) or multigravidae (33%, P <10^-6). Primigravidae showed a higher geometric mean parasite density. In the CQ25 treatment group, failure to clear parasites by day 7 was more common in primigravidae than nulligravidae (P=0.008) or multigravidae (P=0.15). In the CQ5 treatment group, primigravidae were more likely to show increasing parasite density than nulligravidae or multigravidae.     

In vivo Susceptibility of Plasmodium vivax to Chloroquine in Southeastern Iran

Background

Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province, southeastern Iran.

Methods

A total 270 subjects with symptomatic malaria and confirmed P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonucleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0, 5, 6, 7, and 28.

Results

P. vivax was cleared in 15%, 50%, 95%, and 100% of patients on days 1, 2, 3, 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7, 14 and 28. Mean (±standard deviation) parasite clearance time was 2.41 (±0.8) days.

Conclusion

P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compatible with results of neighboring countries Pakistan and Afghanistan.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum

Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (F_ST = 0.253, P < 0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r = 0.49, P = 0.003, N = 83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India.     

Chloroquine-resistant falciparum malaria imported into the Netherlands

A prospective study of imported chloroquine-resistant falciparum malaria in the Netherlands is described. From 1979 to 1983, 77 non-immune patients were investigated; in 41 (53%) decreased sensitivity of Plasmodium falciparum to chloroquine could be confirmed. Signs and symptoms in these patients differ from the classical picture. Resistance to sulfadoxine—pyrimethamine (Fansidar) was established in 6 patients. Parasitaemia was found twice during dapsone—pyrimethamine (Maloprim) prophylaxis. The implications for advice on treatment and prophylaxis are discussed.     

In vivo and in vitro susceptibility to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire

From April to June 1983, combined in vivo and in vitro studies were conducted to assess the response to chloroquine of Plasmodium falciparum in Kinshasa and Mbuji-Mayi, Zaire. A total of 109 patients were treated with chloroquine, either as a single dose of 10 mg/kg or as a full dose of 25 mg/kg. All patients rapidly cleared their asexual parasitaemia, no recurrence being noted during the subsequent 3 weeks of follow-up. In the fourth week, recurrences were noted in 3 out of 66 patients treated with the full dose of chloroquine and in 10 out of 43 patients treated with the single dose. A total of 101 in vitro tests (30 macro tests, 39 micro tests, and 32 48-hour tests) were successfully performed with blood samples collected from 51 of these patients. Full sensitivity to chloroquine was demonstrated in all but 3 of the successful in vitro tests, the results from these 3 tests being contradicted either by alternative in vitro tests or by the corresponding in vivo findings. These investigations thus failed to detect chloroquine resistance at the level reported in East Africa or eastern Zaire (in Kivu).     

In vivo assessment of the sensitivity of Plasmodium falciparum to sulphadoxine/pyrimethamine combination (Fansidar) in six localities in Tanzania where chloroquine-resistant P. falciparum has been detected

The sensitivity of Plasmodium falciparum strains to Fansidar (500 mg sulphadoxine/25 mg pyrimethamine) was tested in vivo in six localities in the United Republic of Tanzania where chloroquine-resistant P. falciparum strains have been demonstrated by both in vivo and in vitro tests. Single doses as recommended by the manufacturers achieved 100% clearance of parasitaemia in five localities with mean clearance period of between 2·2 and 2·9 days. In one locality (Gonja) the recommended dose failed to clear parasitaemia in two of the 38 cases (5·3%) within seven days. The possibility of using this drug combination for the treatment of chloroquine-resistant P. falciparum strains in the United Republic of Tanzania is discussed.     

Response of children with Plasmodium falciparum to chloroquine and development of a national malaria treatment policy in Zaire

In vivo sensitivity of Plasmodium falciparum to chloroquine was evaluated in 4 of 9 regions of Zaire in 1985 to develop a national strategy for treatment of malaria. Children less than 5 years of age were treated with either a single dose of chloroquine base, 10 mg/kg, or a dose of 25 mg/kg given over 3 d. A modified 7-day World Health Organization in vivo test was used with follow-up 2, 3 and 7 d after the start of treatment. 339 children were studied. In Bwamanda 92% of children were aparasitaemic 7 days after chloroquine, 10 mg/kg, but in Kinshasa only 44% were free of parasites after 25 mg/kg chloroquine. The mean drop in parasite density among those who did not clear parasites by day 7 was greater than 98% of the initial value. Although the parasite density decreased markedly, the failure of most subjects to become aparasitaemic indicated a marked decrease in parasite sensitivity since 1983. Only one child of 51 who were initially febrile remained febrile, although 14 (28%) of these had resistant parasites. The decrease in parasitaemia and temperature, even among children with resistant strains, led the Ministry of Health to recommend 25 mg/kg chloroquine as first line treatment for fever/malaria in their national malaria control plan. The plan includes drug sensitivity surveillance and a referral system for patients who do not respond to chloroquine treatment.     

Blood transfusion induced malaria in Iran

Blood transfusion plays the main rôle in induced malaria in Iran. Over 111 cases of transfusion malaria were recorded during the 10 years from 1963 to 1972. Seventy-three% of the species of plasmodia have been P. malariae and 27% P. vivax. 9 cases of transfusion induced quartan malaria in blood recipients have been studied. In 2 blood donors who were proved to be carriers of P. malariae by the fluorescent antibody test, scanty malaria parasites were detected in thick films made from blood concentrate obtained by centrifugation.     

Effect of chlorpheniramine on the pharmacokinetics of and response to chloroquine of Nigerian children with falciparum malaria.

Chlorpheniramine (CP), a histamine H1-receptor antagonist, enhances the efficacy of chloroquine (CQ) in acute uncomplicated falciparum malaria. The effects of this combination therapy on the pharmacokinetic disposition of CQ is, however, unpredictable. A standard treatment with 25 mg CQ base per kg bodyweight was orally administered over 3 days, alone or in combination with CP, to 17 semi-immune Nigerian children with Plasmodium falciparum parasitaemia attending hospital in Lagos, Nigeria, and observed for 28 days. Whole-blood CQ concentrations were monitored 14 times during the follow-up by high-performance liquid chromatography analysis of blood dried on filter paper. Parasitaemia was determined on thick blood films stained with Giemsa, and treatment failures were established following the WHO classification for CQ resistance. Our pharmacokinetic data showed that the peak whole-blood CQ concentration was significantly increased (P < 0.05) by CP administration, and the time to achieve the peak was reduced in the presence of CP. The area under the first-moment drug-concentration-time curve was also significantly increased (P < 0.05) by CP administration. Treatment with CQ-CP combination resulted in a shorter parasite clearance time (2.0 +/- 0.5 days) and a higher cure rate (87.5%) compared to treatment with CQ alone (3.5 +/- 0.5 days; 66.7%). Our data suggest that CP enhanced the efficacy of CQ against resistant P. falciparum in acute uncomplicated malaria by increasing the uptake/concentration of CQ in resistant parasites.