Norfloxacin is inferior to chloroquine for falciparum malaria in northwestern Zambia: a comparative clinical trial.

Fluoroquinolones are active against Plasmodium falciparum in vitro. In a prospective, randomized, comparative trial, norfloxacin, 400 mg twice a day for 3 days, was compared with a standard course of chloroquine in semiimmune adults with symptomatic falciparum malaria in northwestern Zambia, where chloroquine resistance is uncommon. Patients were followed for 28 days. The trial was terminated after 38 patients were studied because chloroquine was markedly more effective, curing all 18 patients (100%) compared with only 8 (40%) of 20 who received norfloxacin (P less than .001). Of the 12 norfloxacin failures, 6 had clearing of trophozoites but recurrence during the study period (RI), 4 had incomplete clearance of trophozoites with later recurrence (RII), and 2 had no improvement (RIII). The mean parasite clearance time was significantly shorter with chloroquine (30.4 vs. 52.7 h; P = .02). The mean defervescence time was also shorter with chloroquine (16.9 vs. 24.5 h; not significant). In contrast to its inferior efficacy, norfloxacin caused fewer adverse effects than did chloroquine (33% vs. 0; P less than .001).     

Problem of antimalarial drug resistance in Plasmodium falciparum in Mizoram.

Studies conducted in Mizoram during 1981 to 1990 have shown areas with increasing RIII level of chloroquine resistance. These foci need urgent liquidation. Sulfalene-pyrimethamine drug combination was found suitable for treatment of P. falciparum cases in these areas with only 5.3 per cent cases showing RI level of Plasmodium response. Quinine combined with sulfalene-pyrimethamine showed 100 per cent success. Amodiaquine however was similar in response to chloroquine though the mean parasite clearance time with amodiaquine was slightly better.     

Immune responses to chloroquine--sensitive and resistant populations of Plasmodium berghei in mice

In order to elucidate the role of the host as a factor in the spread of chloroquine resistance, a study of the host's immune responses in chloroquine resistant (cqr) and chloroquine sensitive (cqs) Plasmodial infections is essential. Course of the infection and the nature of immune responses in mice infected with chloroquine resistant (R) and chloroquine sensitive (S) strains of Plasmodium berghei were compared. Crude parasite antigen activated T cells from both the groups of mice (R and S) and the parasite specific antibodies were detected in the sera of both the groups. The differences in immune responses between the control (uninfected) and infected mice were found to be significant. However, humoral and in vitro cellular responses obtained with T cells from chloroquine resistant P. berghei primed mice was lower in comparison with the responses obtained with T cells from the sensitive infections. Our studies therefore suggest that immunosuppression to parasite antigen is seen in mice primed with chloroquine resistant P. berghei, which may play a role in the development of resistance.     

Evidence that mutant PfCRT facilitates the transmission to mosquitoes of chloroquine-treated Plasmodium gametocytes

Resistance of the human malarial parasite Plasmodium falciparum to the antimalarial drug chloroquine has rapidly spread from several independent origins and is now widely prevalent throughout the majority of malaria-endemic areas. Field studies have suggested that chloroquine-resistant strains might be more infective to mosquito vectors. To test the hypothesis that the primary chloroquine resistance determinant, mutations in PfCRT, facilitates parasite transmission under drug pressure, we have introduced a mutant or wild-type pfcrt allele into the rodent model malarial parasite Plasmodium berghei. Our results show that mutant PfCRT from the chloroquine-resistant 7G8 strain has no effect on asexual blood stage chloroquine susceptibility in vivo or ex vivo but confers a significant selective advantage in competitive mosquito infections in the presence of this drug, by protecting immature gametocytes from its lethal action. Enhanced infectivity to mosquitoes may have been a key factor driving the worldwide spread of mutant pfcrt.     

Sensitivity of Plasmodium vivax to chloroquine in Sa Kaeo Province,Thailand

Following a recent, abrupt local increase in the incidence of vivax malaria, a study was conducted in order to evaluate the efficacy of chloroquine for the treatment of 26 adult patients with acute vivax malaria in Sa Kaeo Province, Thailand. The chloroquine sensitivity of Plasmodium vivax has been assessed in parallel, using a growth inhibition method. Blood samples for the in vitro tests were taken prior to the administration of the standard treatment with chloroquine--in total 25 mg base/kg over 3 days--and primaquine 0.25 mg base/kg once daily for 14 days. The efficacy has been assessed according to the WHO standard in vivo test. The cure rate was 100%. No recrudescence was observed during the follow-up period of 28 days. The mean fever clearance time (FCT) was 40 h, the mean parasite clearance time (PCT) was 49 h. Mean IC(50) and IC(90) of the parasites were 28 and 171 nM, respectively. These results show that local P. vivax is still sensitive to chloroquine. The epidemic outbreak was therefore obviously not due to the presence of chloroquine-resistant P. vivax.     

Isolated malaria outbreak in Somalia: role of chloroquine-resistant Plasmodium falciparum demonstrated in Balcad epidemic

A study was conducted in Balcad in December 1988 during a reported outbreak of malaria in order to investigate possible factors contributing to the outbreak. The slide positivity rate of 71% among fever patients, which was significantly higher than the usual rate, suggests the existence of a fresh malaria outbreak in the area. High parasite densities together with the pronounced malaria symptoms among both resident children and adult immigrants indicated little, if any, malaria experience in these patients. This outbreak could not be explained by gross climatic changes, e.g. unusual rainfalls. The in-vivo response of P. falciparum to the standard therapeutic regimen of chloroquine showed a high degree of resistance, with 31 of 36 patients showing resistance, including five RI responses and 26 RII-RIII responses. Micro in-vitro tests for chloroquine susceptibility showed resistance in 33 out of 37 isolates with a mean EC50 and EC99 of 1.50 and 10.97 X 10(-6) mol l-1 blood, respectively, indicative of a high degree of chloroquine resistance. All isolates tested against sulphadoxine/pyrimethamine showed a sensitive response. The frequent presence (78%) of detectable chloroquine levels prior to treatment did not apparently alter the in-vitro parasite growth. This is a sign of widespread use of this drug resulting in a high 'drug pressure' in the area. The proportion of sensitive parasites was inversely related to the pretreatment chloroquine concentration providing evidence of the selection of resistant parasites by the previous drug intake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium vivax resistance to chloroquine in Dawei, southern Myanmar

Objective  To assess the efficacy of chloroquine in the treatment of Plasmodium vivax malaria in in Dawei District, southern Myanmar.
Methods  Enrolled patients at Sonsinphya clinic >6 months of age were assessed clinically and parasitologically every week for 28 days. To differentiate new infections from recrudescence, we genotyped pre- and post-treatment parasitaemia. Blood chloroquine was measured to confirm resistant strains.
Results  Between December 2002 and April 2003, 2661 patients were screened, of whom 252 were included and 235 analysed. Thirty-four per cent (95% CI: 28.1–40.6) of patients had recurrent parasitaemia and were considered treatment failures. 59.4% of these recurrences were with a different parasite strain. Two (0.8%) patients with recurrences on day 14 had chloroquine concentrations above the threshold of 100 ng/ml and were considered infected with chloroquine resistant parasites. 21% of failures occurred during the first 3 weeks of follow-up: early recurrence and median levels of blood chloroquine comparable to those of controls suggested P. vivax resistance.
Conclusions  Plasmodium vivax resistance to chloroquine seems to be emerging in Dawei, near the Thai-Burmese border. While chloroquine remains the first-line drug for P. vivax infections in this area of Myanmar, regular monitoring is needed to detect further development of parasite resistance.     

Therapeutic responses to antimalarial and antibacterial drugs in vivax malaria

Plasmodium vivax is the most prevalent malaria infection and is an important cause of morbidity in Central and South America and Asia. P. vivax is generally sensitive to the common antimalarial drugs but high level resistance to chloroquine and/or pyrimethamine has been documented in some geographic locations. In the studies reviewed here, the therapeutic responses to antimalarial and antibacterial drugs in vivax malaria have been assessed in the Bangkok Hospital for Tropical Diseases. The evaluated drugs consisted of the eight most widely used antimalarial drugs and anti-bacterial drugs that possess antimalarial activities (tetracycline, doxycycline, clindamycin or azithromycin). The activities of these drugs in descending order of parasite clearance times were artesunate, artemether, chloroquine, mefloquine, quinine, halofantrine, primaquine, followed by the antibacterial drugs and lastly sulfadoxine-pyrimethamine. Clinical responses to sulfadoxine-pyrimethamine were also poor with evidence of high grade resistance in 42% of the patients. Of the four antibacterial drugs, clindamycin was more effective than azithromycin and can be an alternative to the tetracyclines. Except for chloroquine and mefloquine which have long plasma half lives and may therefore suppress first relapses, the cumulative cure rates for the short acting antimalarial drugs were similar. Double infection with Plasmodium falciparum was common and usually manifested 3-4 weeks following clearance of vivax malaria. The prevalence of cryptic falciparum malaria was 8-15% and was higher in patients treated with less potent antimalarial drugs. Follow-up studies have revealed that the relapse time in Thai patients with vivax malaria is on average only 3 weeks, but can be suppressed by the slowly eliminated antimalarial drugs such as chloroquine and mefloquine. For accurate comparison of relapse/recrudescence rates in vivax malaria, at least 2 month's follow-up is required. It can be concluded that in malarious areas of Thailand, double infection with P. falciparum and P. vivax is common affecting at least 25% of the patients and usually manifests as sequential illnesses. P. vivax in Thailand is sensitive to chloroquine but has acquired high grade resistance to sulfadoxine-pyrimethamine.     

Antimalarial drug susceptibility testing of Plasmodium falciparum in Thailand using a microdilution radioisotope method

Antimalarial activity of chloroquine, quinine, mefloquine and halofantrine against 33 strains of P. falciparum isolated from naturally acquired malaria infections in Thailand was determined using a radioisotope microdilution method. A microtitration procedure was used to test isolates of P. falciparum against the 4 drugs simultaneously. The mean ID50 for chloroquine and quinine reflected known resistance to those drugs in Thailand. The mean ID50 for mefloquine and halofantrine showed susceptibility to these drugs. Four isolates of P. falciparum however had markedly decreased susceptibility to mefloquine (ID50 greater than 15 ng/ml); one case of which was confirmed as the first case of RII resistance for mefloquine in Thailand. Several parasite isolates were also observed to have decreased susceptibility to the new drug, halofantrine. These studies strongly recommend that in vitro testing be done in conjunction with field evaluation of new antimalarial drugs.     

Micro in vitro assessment of Plasmodium falciparum sensitivity to chloroquine and mefloquine in Gujarat

Micro in vitro tests conducted in 1987 in Surat district of Gujarat on sensitivity status of P. falciparum to chloroquine and mefloquine revealed that the parasite has developed resistance to chloroquine upto 32 pmol. The ED 99 in Hazira, Gothan and Umra areas of the district was found to be 17.3, 18.5 and 8.7 pmol/well for chloroquine and for mefloquine it was 14.5, 4.8 and 6.8 pmol/well respectively. Monitoring of P. falciparum resistance is indicated under National Malaria Eradication Programme.     

Atypical manifestations of Plasmodium vivax malaria

About 110 patients were enrolled to study the atypical presentations and the chloroquine sensitivity pattern of Plasmodium vivax malaria. The diagnosis was made from Giemsa stained peripheral blood smear. The co-infection of falciparum malaria was excluded both by smear and ParaSight F-test. After a thorough clinical work up, biochemical investigations were done. The fever clearance and parasite clearance time were determined in all cases. Absence of malarial paroxysm (22.8 per cent), migrainous headache (4.5 per cent), myalgia (6.3 per cent), episodic urticarial rash (1.8 per cent), relative bradycardia (13.6 per cent) and postural hypotension (2.7 per cent) were the atypical manifestations encountered. Besides this, severe forms like jaundice (7.2 per cent), cerebral involvement (0.9 per cent), severe anaemia (7.2 per cent), thrombocytopenia (3.6 per cent) and pancytopenia (0.9 per cent) had been detected. All, except the patient with cerebral involvement were treated with chloroquine patients responded well to the treatment except two (1.8 per cent) patients who had chloroquine resistance. This study showed that vivax malaria can present with atypical and protean manifestations. The changing clinical profile along with development of chloroquine resistance may be considered as a warning signal.     

Biochemical aspects of drug action and resistance in malaria parasites

Biochemical aspects of action of antifolates and 4-aminoquinolines and their resistance in the malaria parasites are reviewed, with emphasis on pyrimethamine and chloroquine respectively. Resistance to pyrimethamine has been shown to be associated with either an increase in the amount of parasite dihydrofolate reductase or a reduced affinity of the enzyme for drug binding, in line with the presence of a distinctive pathway for folate metabolism. The theories for drug synergism in the folate pathway are discussed with respect to resistance to pyrimethamine and its combination with sulpha drugs. The biochemical basis for chloroquine resistance is still unclear, reflecting incomplete understanding of its mechanism of action. Data implicating the role of haemozoin and other components as a putative chloroquine receptor of the parasites are reviewed, and possible explanations for resistance are discussed.     

Antipyretic, parasitologic, and immunologic effects of combining sulfadoxine/pyrimethamine with chloroquine or paracetamol for treating uncomplicated Plasmodium falciparum malaria

Sulfadoxine/pyrimethamine (SP) is increasingly used against malaria in sub-Saharan Africa because of chloroquine resistance. However, chloroquine may have a beneficial antipyretic effect. We therefore compared the combination of SP plus chloroquine, chloroquine alone, SP alone, and SP plus paracetamol in the treatment of uncomplicated Plasmodium falciparum malaria in 175 Tanzanian children (1-4 years old) in a randomized trial. Outcome variables were axillary temperatures every six hours, daily parasitemias, and serum levels of IgG antibodies to P. falciparum. Lower mean temperatures (6-48 hours) were achieved with SP plus chloroquine or paracetamol than with SP alone (P < 0.001) or chloroquine alone (P < 0.05). All three SP-treated groups showed high and similar parasite reduction (0-48 hours), whereas treatment with chloroquine alone was much less effective. Levels of IgG antibodies to P. falciparum increased significantly (P < 0.001) and similarly in the four treatment groups between days 0, 2, and 3. Thus, the addition of chloroquine or paracetamol to SP improved the clinical outcome, but did not affect the parasitologic response or antibody production.     

Response of Kampuchean strains of Plasmodium falciparum to antimalarials: in-vivo assessment of quinine and quinine plus tetracycline; multiple drug resistance in vitro

Forty-three patients were enlisted in the in-vivo test for sensitivity of Kampuchean strains of Plasmodium falciparum to quinine. The mean parasite density count on day 0 was 32,398 asexual parasites per microliter of blood. With a dosage of 1.5 g quinine base daily for 10 days the average parasite clearance time was 5.6 days, and the average duration of fever 3.4 days. The in-vivo test was evaluated at 7 and 10 days after the start of therapy. After 7 days only 16 patients were parasite negative by microscopic examination (S); 20 patients had an RI recrudescence, four patients responded at the RII level and three at the RIII level. Evaluating the in-vivo test at 10 days, the number of patients parasite negative increased to 18, the number of those with an RI level of resistance increased to 21, two patients gave an RII response and two had an RIII level of resistance. Twenty-two adult males were included in an in-vivo test of the sensitivity of P. falciparum to quinine plus tetracycline. The course of treatment was: quinine 3 x 500 mg daily for 10 days, tetracycline 3 x 500 mg for 7 days. Parasite density counts on day 0 averaged 11,393 asexual parasites per microliter of blood. The average parasite clearance time was 5.9 days, and the average duration of fever was 3.8 days. After 7 days of treatment, 81.8% of patients were parasite negative, while one patient had a recrudescence after 17 days (RI). Three infections were resistant at the RII level. By prolongation of the observations to day 10, the parasitaemia was cleared in all patients, i.e. all infections were sensitive to quinine plus tetracycline. Thirty-four patients with falciparum malaria were screened for in-vitro resistance to chloroquine, mefloquine and quinine using the WHO standard micro-test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic and genotypic characteristics of recently adapted isolates of Plasmodium falciparum from Thailand.

The drug sensitivity characteristics and Plasmodium falciparum pfmdr1 status of five isolates of P. falciparum recently isolated from patients presenting for treatment from the Thailand/Myanmar border have been investigated. The aim of the study was to avoid the criticisms of some earlier studies by focusing on newly collected isolates from a specific geographic location. Three of the isolates studied exhibited clear resistance to chloroquine similar to that observed in the K1 Thai standard isolate obtained in the 1970s, and the other two isolates were of intermediate sensitivity to chloroquine with concentrations of drug that inhibit parasite growth by 50% of 50 and 43 nmol. The sensitivity of all isolates was enhanced by verapamil but we found no clear association between chloroquine sensitivity and gene copy number or intra-allelic variation of pfmdr1. In contrast, clear cross-resistance was seen between mefloquine and halofantrine, with the most sensitive isolates carrying the K1 mutation in pfmdr1.     

Plasmodium falciparum sensitivity to erythromycin and 4-aminoquinoline combinations in vitro.

Although erythromycin has been reported to be active against Plasmodium falciparum in vitro and P. berghei in vivo and in vitro when given alone or with chloroquine, it has been difficult to demonstrate a beneficial effect for the combination of erythromycin and chloroquine when used for the treatment of P. falciparum infections in humans. We developed a seven-day test of parasite sensitivity to a 4-aminoquinoline and erythromycin combination in vitro. Eight isolates of P. falciparum from the Kenyan coast were culture-adapted and exposed to erythromycin with chloroquine or with amodiaquine. The interaction of the drugs was evaluated by plotting the concentration of each drug needed to inhibit parasite growth. In seven isolates the combination of chloroquine and erythromycin was antagonistic; one isolate showed slight synergy The combination of amodiaquine and erythromycin was synergistic in three isolates but antagonistic in five. An antagonistic interaction may explain why erythromycin does not enhance chloroquine treatment of malaria in vivo in Kenya.     

Serial studies on the evolution of drug resistance in malaria in an area of east Africa: findings from 1979 up to 1986

Observations, previously reported for 1979-82, have been continued up to 1986 on the development of drug resistance in P. falciparum in the North Mara area of Tanzania, where a chloroquine chemosuppression campaign was attempted from 1977 to 1982. The WHO micro in-vitro test for chloroquine and other drugs was used. Because of the large number of tests done, each test was characterized by the mean minimum inhibitory concentrations (MIC) of drug needed to prevent schizont development instead of counting the numbers of schizonts. The MIC for chloroquine has risen progressively each year but changes in the findings of in-vivo tests were less dramatic possibly due to the effects of immunity. Resistance to amodiaquine has followed that to chloroquine at a lower level, and in the last years the MIC for quinine has risen. Sporadic resistance to mefloquine was found and, by in-vivo test, to sulphadoxine-pyrimethamine. Possible factors in the evolution of drug resistance are discussed together with implications for the future.     

An in vitro system for assessing the sensitivity of Plasmodium vivax to chloroquine

Following earlier observations on short-term culture of Plasmodium vivax, an in vitro test system has been developed for assessing the parasite's sensitivity to chloroquine. Fresh isolates with predominantly young trophozoites are diluted 1:19 with a (v/v=1/1) mixture of RPMI 1640 and Waymouth medium. The blood-medium mixture (BMM) is inoculated into the predosed microtitre plates before incubation in a candle jar. Incubation for 30 or 42 h yielded the best results. Incubation for 18 or 24 h was generally insufficient for an adequate development of the parasites. The reading of the test is based on stage-specific differential counts in the Giemsa-stained pre-incubation and post-incubation thick films, the evaluation on log-probit analysis of drug-related inhibition of parasite development. The test system has been evaluated on 200 fresh P. vivax isolates in an area with satisfactory clinical-parasitological response to chloroquine. At 30 or 42 h incubation 121 isolates (61.5%) showed adequate control growth and yielded valid sensitivity tests. Complete inhibition of parasite development occurred within the concentration range of 40-1280 nM. The mean EC50 for 30 h of incubation was 50.3 nM, as compared to 49.7 nM with 42 h of incubation. The geometric mean cut-off concentration of parasite development was 488 nM with 30 h of incubation as against 470 nM with 42 h of incubation.     

Artemisinin or chloroquine for blood stage Plasmodium vivax malaria in Vietnam

Chloroquine-resistant Plasmodium vivax has not yet occurred in Vietnam. The efficacy of artemisinin for P. vivax was not established. We conducted a double-blind randomized study involving 240 inpatients with P. vivax malaria who received artemisinin (40 mg/kg over 3 days) plus placebo chloroquine (Art) or chloroquine (25 mg/kg over 3 days) plus placebo artemisinin (Chl). Patients were followed up with weekly blood smears for 28 days. In each group 113 cases were analysed. All patients recovered rapidly. The median (range) parasite clearance time of regimen Art was 24 h (8-72) and of Chl 24 h (8-64; P = 0.3). Parasites reappeared in two cases in each group on day 14, in eight cases in each group (7%) on day 16 and in 25 (23%) and 18 (16%) cases, respectively, at the end of 4-week follow-up (P = 0.3). The population parasite clearance curve followed a mono-exponential decline. The parasite reduction ratio per 48 h reproduction cycle was 2.3 x 104 for both regimens. We conclude that artemisinin and chloroquine are equally effective in the treatment of P. vivax infections in Vietnam. Reappearance of parasites before day 16 (7%) suggests the emergence of chloroquine resistance. Three days of artemisinin monotherapy does not prevent recrudescence.     

Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine.

Two chloroquine-resistant cloned isolates of Plasmodium falciparum were subjected to mefloquine selection to test if this resulted in alterations in chloroquine sensitivity and amplification of the pfmdr1 gene. The mefloquine-resistant lines derived by this selection were shown to have amplified and overexpressed the pfmdr1 gene and its protein product (Pgh1). Macrorestriction maps of chromosome 5, where pfmdr1 is encoded, showed that this chromosome has increased in size in response to mefloquine selection, indicating the presence of a gene(s) in this area of the genome that confers a selective advantage in the presence of mefloquine. Concomitant with the increase in mefloquine resistance was a corresponding increase in the level of resistance to halofantrine and quinine, suggesting a true multidrug-resistance phenotype. The mefloquine-selected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased pfmdr1 gene copy number. These results have important implications for the derivation of amplified copies of the pfmdr1 gene in field isolates, as they suggest that quinine pressure may be involved.