In Vitro and In Vivo Antimalarial Activity of Ficus thonningii Blume (Moraceae) and Lophira alata Banks (Ochnaceae), Identified from the Ethnomedicine of the Nigerian Middle Belt.

Drug resistance in Plasmodium falciparum requires that new drugs must be developed. Plants are a potential source for drug discovery and development. Two plants that used to treat febrile illnesses in Nigeria were tested for in vitro and in vivo antimalarial activity and cytotoxicity in cancer cell lines. Methanol, hexane, and ethyl acetate leaf extracts of Ficus thonningii and Lophira alata were active in in vitro assays against P. falciparum NF54 (sensitive) and K1 (multiresistant) strains. Hexane extracts of F. thonningii and L. alata were the most effective extracts in in vitro assays with IC50 of 2.7 ± 1.6 µg/mL and 2.5 ± 0.3 µg/mL for NF54 and 10.4 ± 1.6 µg/mL and 2.5 ± 2.1 µg/mL for K1 strain. All extracts were nontoxic in cytotoxicity assays against KB human cell line with IC50 of over 20 µg/mL, demonstrating selectivity against P. falciparum. In vivo analysis shows that hexane extracts of both plants reduced parasitaemia. At the maximum dose tested, L. alata had a 74.4% reduction of parasitaemia while F. thonningii had a reduction of 84.5%, both extracts prolonged animal survival in mice infected with P. berghei NK65 when compared with vehicle treated controls. The antiplasmodial activity observed justifies the use of both plants in treating febrile conditions.

Assessment and characterization of Ca2+-ATPase expression in selected isolates and clones of Plasmodium falciparum.

Ca2+-ATPase expression in 15 selected isolates from malaria patients at the University College Hospital (UCH) Ibadan and two cloned strains (W2-chloroquine resistant, D6-chloroquine sensitive) of P.falciparum was assessed using spectrophotometric assay method. The kinetics of activity of Ca2+- ATPase in three isolates (NCP 14, NCP5, NCP1) and two clones (W2, D6) also assessed. 12% SDS-PAGE analysis of total proteins in one isolate (NCP14) and two clones (W2, D6) was also investigated. All the selected isolates and the two cloned strains exhibited measurable Ca2+-ATPase activity. The Ca2+-ATPase activity in cloned strain D6 (6.50 + 0.74mmolPi/min/mg protein) was higher than in cloned strain W2 (3.93 + 0.61mmolPi/min/mg protein. The Ca2+-ATPase activity in isolates from malaria patients varied widely (1.95 + 0.74 - 21.56 +1.43mmolPi/min/mg protein). The kinetic constants obtained for the two cloned strains showed that clone W2 had a higher Vmax (Vmax = 363mmolPi/min/mg protein) than clone D6 (Vmax = 74mmolPi/min/mg protein). All the isolates and the two cloned strains showed similar affinity for ATP (Km ~ 10mM). Scan of SDS-PAGE gel of total proteins in the isolate and cloned strains showed the presence of oligopeptide bands of molecular weights range of 148-176 kDa; 116-123 kDa respectively. These suggest the presence of predicted polypeptide of Ca2+-ATPase nature of molecular weight estimate of 139 kDa. The study agrees with previous findings that Ca2+-ATPase is functionally expressed in P.falciparum, The study also indicates that Ca2+-ATPase functional expression may vary with isolate or clone but the ATP binding mechanism to the enzyme is similar in all isolates and clones of P. falciparum. The study further suggests a possible association between acquisition of chloroquine resistance and Ca2+-ATPase functional expression in P. falciparum.

Effect of chloroquine phosphate and toxic concentrations of lead acetate on Ca2+-ATPase activity in isolates and clones of Plasmodium falciparum.

The basal activity of Ca2+-ATPase in two isolates (NL56, UNC) and two clones (D6, W2) of P.falciparum was assessed. The effects of various concentrations of chloroquine phosphate and toxic concentrations of lead acetate were also evaluated in the clones and strains of P.falciparum. The Ca2+-ATPase activity was measured by monitoring the rate of release of inorganic phosphate from the gamma-position of ATP on spectrophotometer at 820nm wavelength. The various concentrations of chloroquine (3, 6, 9, 12, 18µg/ml) and lead acetate (5, 10, 20, 30, 40µg/ml) on Ca2+-ATPase activity were measured respectively. Chloroquine phosphate inhibited Ca2+-ATPase activity in both the isolates and the cloned strains of P.falciparum in concentration dependent manner. Median Inhibitory concentration of chloroquine (MIC50) estimated from the plot of activity against chloroquine concentration was found to be 2.6mg/ml at pH 7.4 for both the isolates and cloned strains examined. Lead acetate at concentrations 5-20µg/ml inhibited Ca2+-ATPase activity in concentration dependent manner in clone W2 (Chloroquine resistant strain) while the same range of concentrations of lead acetate stimulated the activity of the enzyme in clone D6 (Chloroquine sensitive strain).The inhibitory effect of lead acetate on the enzyme in clone D6 was observed at concentrations above 20µg/ml. The result also suggests that lead ions could modulate and moderate calcium ion homeostasis in P. falciparum via its effect on Ca2+-ATPase activity. Also sufficient influx of lead ions into P. falciparum may transform the biochemical or bioenergetics nature of chloroquine sensitive strain of P. falciparum (D6) to that similar to chloroquine resistant strain (W2). In conclusion, inhibition of Ca2+-ATPase activity of P.falciparum may be part of the mechanism of action of chloroquine in its use as chemotherapy for malaria. The study implies that populations simultaneously exposed to lead pollution and malaria infection may experience failure in chloroquine therapy.

Community effectiveness of artemisinin-based combination therapy for malaria in rural southwestern Nigeria.

A descriptive cross sectional survey using an interviewer-administered questionnaire was carried out among 700 caregivers whose children had fever during the previous two weeks. The aim was to determine the community effectiveness of malaria treatment using arthemeter-lumefantrine (AL) among under-5-year-olds in a rural community in southwestern Nigeria. A total of 353 (50.9%) children received AL. About half of these children (49%) were said to have been treated within 24 hours of onset of symptoms; 44% took the drug for the stipulated period of time; 42% received the correct dosage; and only 4% received all the treatment steps. With a drug efficacy of 100%, AL achieved a community effectiveness of 4%. The greatest effort in the home management of malaria strategy should be in reducing delay in treatment and improving dosage and duration of treatment.

Chloroquine Resistant Plasmodium falciparum in Nigeria: Relationship between pfcrt and pfmdr1 Polymorphisms, In-Vitro Resistance and Treatment Outcome.

This study was designed to evaluate the association between polymorphisms in pfcrt and pfmdr1 genes and in-vitro chloroquine (CQ) sensitivity in fresh isolates of P. falciparum and patients' treatment outcome. The modified schizont inhibition assay was used to determine in-vitro sensitivity of P. falciparum. Polymorphisms in pfcrt and pfmdr1 genes were detected using nested PCR and RFLP techniques in 84 P. falciparum isolates obtained from patients with acute uncomplicated malaria.Eighty five percent (71/84) and 15% (13/84) of the parasites were resistant and sensitive in-vitro to CQ respectively. Molecular analysis showed presence of mutant pfcrtT76, pfmdr1Y86 and pfmdr1F184 alleles in 60%, 33% and 14% of the isolates respectively. There was a significant association between in-vitro and in-vivo CQ resistance (p=0.029) and also between the presence of mutant pfcrtT76+pfmdr1 Y86-Y184 haplotype and in-vitro (p=0.013) or in-vivo CQ resistance (p=0.024).Overall results from this study demonstrates that the presence of pfcrtT76+ pfmdr1 Y86-Y184 haplotype in Nigerian isolates of Plasmodium falciparum is predictive of in-vitro and in-vivo CQ resistance and therefore may be useful for monitoring resistance to this drug.

Effects of pyrimethamine-sulphadoxine, chloroquine plus chlorpheniramine, and amodiaquine plus pyrimethamine-sulphadoxine on gametocytes during and after treatment of acute, uncomplicated malaria in children

The effects of pyrimethamine-sulphadoxine (PS), chloroquine plus chlorpheniramine, a H1 receptor antagonist that reverses chloroquine resistance in Plasmodium falciparum in vitro and in vivo (CQCP), and amodiaquine plus pyrimethamine-sulphadoxine (AQPS) on gametocyte production were evaluated in 157 children with acute, symptomatic, uncomplicated falciparum malaria who were treated with these drugs. PS was significantly less effective than CQCP or AQPS at clearing asexual parasitaemia or other symptoms of malaria. Gametocyte carriage on days 3, 7, and 14 were significantly higher in those treated with PS. The ratio of the density (per æl blood) of peripheral young gametocyte (PYG), that is, < stage III to peripheral mature gametocyte (PMG), that is, stage IV and V, an index of continuing generation of gametocytes, rose to 1 by day 7 of treatment in those treated with PS, but remained consistently below 1 in the other treatment groups. PYG-PMG density ratio increased significantly from day 0-14 in those treated with PS and CQCP (chi2 = 76, P = 0.000001 and chi2 = 42.2, P = 0.00001, respectively) but decreased significantly in those treated with AQPS (chi2 = 53.2, P = 0.000001). Both PS-sensitive and -resistant infections generated PYG (18 of 29 vs 13 of 20, chi2 = 0.04, P = 0.93) but PYG was present only in those with resistant response to CQCP. Combination of PS with amodiaquine (AQ), that is, (AQPS) resulted in less production of PYG, but in this setting, PYG was not indicative of response to AQPS. These data indicate that PS enhanced production or release of young gametocytes when used alone, but generated less young gametocytes when used in combination with AQ. PYG may be used as an indicator of response to CQCP but not PS or PS-based combination drugs.

Predictors of the failure of treatment with pyrimethamine-sulfadoxine in children with uncomplicated falciparum malaria.

The prevalence of pyrimethamine-sulfadoxine (PS)-resistant Plasmodium falciparum malaria has been increasing in sub-Saharan Africa or other parts of the world in the last one or two decades. The factors that identify children at risk of treatment failure after being given PS were evaluated in 291 children with acute, symptomatic, uncomplicated, P. falciparum malaria. The children took part in four antimalarial drug trials between July 1996 and July 2004 in a hyperendemic area of southwestern Nigeria. Following treatment, 64 (22%) of 291 children failed treatment by day 7 or 14. In a multivariate analysis, an age < or = 1.5 years (AOR=2.9, 95% CI 1.3-6.4, P = 0.009) and presence of fever (AOR = 3.3, 95% CI 1.28-7.14, P = 0.01) were independent predictors of the failure of treatment with PS at presentation. Following treatment, delay in parasite clearance >3 days (AOR = 2.56, CI 1.19-5.56, P = 0.016) was an independent predictor of the failure of treatment with PS. In addition, compared with the children who had no fever then, children with fever three or more days after starting treatment were more likely to be treatment failures. These findings may have implications for malaria control efforts in some sub-Saharan African countries where treatment of malaria disease depends almost entirely on PS monotherapy, and for programmes employing PS or PS-based combination therapy.

Effects of pyrimethamine-sulphadoxine, chloroquine plus chlorpheniramine, and amodiaquine plus pyrimethamine-sulphadoxine on gametocytes during and after treatment of acute, uncomplicated malaria in children.

The effects of pyrimethamine-sulphadoxine (PS), chloroquine plus chlorpheniramine, a H1 receptor antagonist that reverses chloroquine resistance in Plasmodium falciparum in vitro and in vivo (CQCP), and amodiaquine plus pyrimethamine-sulphadoxine (AQPS) on gametocyte production were evaluated in 157 children with acute, symptomatic, uncomplicated falciparum malaria who were treated with these drugs. PS was significantly less effective than CQCP or AQPS at clearing asexual parasitaemia or other symptoms of malaria. Gametocyte carriage on days 3, 7, and 14 were significantly higher in those treated with PS. The ratio of the density (per microl blood) of peripheral young gametocyte (PYG), that is, < or = stage III to peripheral mature gametocyte (PMG), that is, stage IV and V, an index of continuing generation of gametocytes, rose to 1 by day 7 of treatment in those treated with PS, but remained consistently below 1 in the other treatment groups. PYG-PMG density ratio increased significantly from day 0-14 in those treated with PS and CQCP (chi2 = 76, P = 0.000001 and chi2 = 42.2, P = 0.00001, respectively) but decreased significantly in those treated with AQPS (chi2 = 53.2, P = 0.000001). Both PS-sensitive and -resistant infections generated PYG (18 of 29 vs 13 of 20, chi2 = 0.04, P = 0.93) but PYG was present only in those with resistant response to CQCP. Combination of PS with amodiaquine (AQ), that is, (AQPS) resulted in less production of PYG, but in this setting, PYG was not indicative of response to AQPS. These data indicate that PS enhanced production or release of young gametocytes when used alone, but generated less young gametocytes when used in combination with AQ. PYG may be used as an indicator of response to CQCP but not PS or PS-based combination drugs.

Open randomized study of artesunate-amodiaquine vs. chloroquine-pyrimethamine-sulfadoxine for the treatment of uncomplicated Plasmodium falciparum malaria in Nigerian children.

BACKGROUND: Artemisinin-based combination antimalarials are currently considered effective alternatives for the treatment of malaria in Africa, but there are few studies of such combinations in Nigerian children. We assessed the safety, treatment efficacy and effects on gametocyte carriage of the combination of artesunate plus amodiaquine and chloroquine plus pyrimethamine-sulfadoxine in children. METHODS: We evaluated 153 children who were aged 12 years or younger who had uncomplicated Plasmodium falciparum malaria. Patients were randomly assigned a combination of artesunate (4 mg/kg of body weight daily for 3 days) plus amodiaquine (30 mg/kg over 3 days), or chloroquine (25 mg/kg over 3 days) plus pyrimethamine-sulfadoxine (25 mg/kg of the sulfadoxine component at presentation). The primary endpoints were the proportions of children with adequate clinical and parasitological response, late parasitological failure, late clinical failure and early treatment failure. The parasitological cure rates on days 14-28 were also used as the primary endpoints. RESULTS: Both regimens were well tolerated; no child was withdrawn because of drug intolerance. All children treated with artesunate plus amodiaquine had adequate clinical and parasitological response (ACPR), while all but five children treated with chloroquine plus pyrimethamine-sulfadoxine had similar response. Fever clearance times were similar in the two treatment groups. However, the proportion of patients whose parasitaemia cleared by day 2 was significantly higher (100 vs. 50%, P = 0.00001) and parasite clearance was significantly faster (1.7 +/- 0.4 vs. 2.5 +/- 0.8 days, P = 0.0001) in children treated with artesunate plus amodiaquine. The cure rates on days 21 (100%vs. 94%, P = 0.03) and 28 (100%vs. 90%, P = 0.003) were also significantly higher in children treated with artesunate plus amodiaquine than in those treated with chloroquine plus pyrimethamine-sulfadoxine. Overall, a significantly higher proportion of children treated with chloroquine plus pyrimethamine-sulfadoxine carried gametocytes at least once during follow-up compared with those treated with artesunate plus amodiaquine [5 of 50 (10%) vs. 1 of 103 (0.97%), P = 0.01]. CONCLUSION: The combination of artesunate plus amodiaquine is therapeutically superior to a combination of chloroquine plus pyrimethamine-sulfadoxine, and significantly reduced gametocyte carriage following treatment.

Predictors of the failure of treatment with chloroquine in children with acute, uncomplicated, Plasmodium falciparum malaria, in an area with high and increasing incidences of chloroquine resistance.

Resistance to chloroquine (CQ) in Plasmodium falciparum has reached unacceptably high levels in many endemic countries. The pre-treatment factors that identify the children who are at risk of treatment failure after being given CQ were evaluated in 385 children with acute, uncomplicated, Plasmodium falciparum malaria. These children each took part in one of six antimalarial drug trials conducted, between July 1996 and July 2004, in a hyper-endemic area of south-western Nigeria. Following treatment with CQ, 149 (39%) of the children failed treatment by day 7 or 14. In a multivariate analysis, an age of < or =7 years [giving an adjusted odds ratio (AOR) of 2.17, with a 95% confidence interval (CI) of 1.19-3.85; P = 0.01], an asexual parasitaemia of > or =100,000/microl (AOR = 2.17; CI = 1.08-4.35; P = 0.03), the presence of gametocytaemia (AOR = 2.08; CI = 1.14-3.85; P = 0.02) and enrolment >4 years after commencement of the study (i.e. after 2000; AOR = 2.13; CI = 1.3-4.0; P = 0.003) were found to be independent predictors at presentation of the subsequent failure of treatment with CQ. Compared with the other children, those who failed to clear their parasitaemias within 3 days and those who still had fever 1-2 days after commencing treatment were more likely to be treatment failures. Together, these findings may have implications for malaria-control efforts in all areas of sub-Saharan Africa where treatment of malaria depends almost entirely on antimalarial monotherapy.

Effects of antifolates--co-trimoxazole and pyrimethamine-sulfadoxine--on gametocytes in children with acute, symptomatic, uncomplicated, Plasmodium falciparum malaria.

Antimalarial drugs including the antifolate, pyrimethamine-sulfadoxine (PS), can modulate the prevalence and intensities of gametocytaemia following treatment of acute malaria infections. They may also directly influence the transmission and spread of drug insensitivity. Little is known of the effects of co-trimoxazole (Co-T), another antifolate antimalarial, on gametocytes in children with acute malaria infections. We compared the effects of Co-T and PS on the prevalence and intensities of gametocytaemia and gametocyte sex ratios in 102 children aged 0.5-12 years presenting with acute and uncomplicated falciparum malaria. Compared to pre-treatment, both drugs significantly increased gametocyte carriage post-initiation of treatment. However, gametocyte carriage was significantly lower on day 14 in those treated with Co-T than PS. Significant increase in gametocytaemia with time occurred in PS--but not Co-T-treated children. Kaplan-Meier survival curve of the cumulative probability of remaining gametocyte-free in children who were agametocytaemic at enrollment showed that by day 7 of follow up, children treated with PS had a significantly higher propensity to have developed gametocytes than in Co-T-treated children (Log-rank statistic 5.35, df = 1, P = 0.02). Gametocyte sex ratio changes were similar following treatment with both drugs. PS and Co-T treatment of acute malaria infections in children from this endemic area is associated with significant increases in prevalence and intensities of gametocytaemia but these effects are more marked in those treated with PS than Co-T.

Effects of antifolates - co-trimoxazole and pyrimethamine-sulfadoxine - on gametocytes in children with acute, symptomatic, uncomplicated, Plasmodium falciparum malaria

Antimalarial drugs including the antifolate, pyrimethamine-sulfadoxine (PS), can modulate the prevalence and intensities of gametocytaemia following treatment of acute malaria infections. They may also directly influence the transmission and spread of drug insensitivity. Little is known of the effects of co-trimoxazole (Co-T), another antifolate antimalarial, on gametocytes in children with acute malaria infections. We compared the effects of Co-T and PS on the prevalence and intensities of gametocytaemia and gametocyte sex ratios in 102 children aged 0.5-12 years presenting with acute and uncomplicated falciparum malaria. Compared to pre-treatment, both drugs significantly increased gametocyte carriage post-initiation of treatment. However, gametocyte carriage was significantly lower on day 14 in those treated with Co-T than PS. Significant increase in gametocytaemia with time occurred in PS - but not Co-T-treated children. Kaplan-Meier survival curve of the cumulative probability of remaining gametocyte-free in children who were agametocytaemic at enrolment showed that by day 7 of follow up, children treated with PS had a significantly higher propensity to have developed gametocytes than in Co-T-treated children (Log-rank statistic 5.35, df = 1, P = 0.02). Gametocyte sex ratio changes were similar following treatment with both drugs. PS and Co-T treatment of acute malaria infections in children from this endemic area is associated with significant increases in prevalence and intensities of gametocytaemia but these effects are more marked in those treated with PS than Co-T.

Plasmodium falciparum malaria in Nigerian children during high and low transmission seasons: gametocyte carriage and response to oral chloroquine.

Plasmodium falciparum malaria during high and low transmission seasons was evaluated in 1031 children treated with different antimalarial drug in a hyperendemic area of southwestern Nigeria. Seventy-three (10.5%) of 693 and forty (11.8%) of 338 children were gametocyte carriers in the high transmission seasons (HTS) and low transmission seasons (LTS), respectively. In a multiple regression model, two factors were found to be independent risk factors for the presence of gametocytemia at enrolment in the HTS: duration of illness >3 d, and asexual parasite densities less than 10,000/microl. Similarly male gender, duration of illness >4 d and parasite density less than 5000/mul were found independent risk factors for presence of gametocytemia during LTS. The presenting parasitemia, parasite clearance times, intensity of gametocytemia and proportion carrying gametocytes post treatment differ significantly in the 333 (32.3%) of these children that were treated with chloroquine in the two seasons. These findings may be important in our understanding of P. falciparum transmission sustenance, response to chloroquine therapy and contribution of chloroquine to gametocyte carriage as seasonal changes occur.

Predictors of the failure of treatment with chloroquine plus chlorpheniramine, in children with acute, uncomplicated, Plasmodium falciparum malaria.

Resistance to chloroquine in Plasmodium falciparum can be reversed, both in vitro and in vivo, by chlorpheniramine, a histamine H(1) receptor antagonist. This reversal raises the possibility of using chlorpheniramine to prolong the clinical usefulness of chloroquine in resource-poor communities. The factors that identify children at risk of treatment failure after being given chloroquine plus chlorpheniramine have now been evaluated in 281 children with uncomplicated, P. falciparum malaria. The children, who had taken part in six trials of antimalarial drugs between February 1996 and September 1999, in a hyper-endemic area of south-western Nigeria, were enrolled prospectively for the present study. Following treatment with chloroquine plus chlorpheniramine, 13 (5%) of the children failed treatment by day 7 or 14. In a multivariate analysis, an age of < or =3 years (adjusted odds ratio = 11.1; 95% confidence interval = 2.2-55.3; P = 0.003) and a parasitaemia that took >3 days to clear (adjusted odds ratio=7.9; 95% confidence interval = 1.3-49.4; P = 0.027) were found to be independent predictors of treatment failure. In addition, compared with the children who had a lower axillary temperature then, the children who had an axillary temperature of > or =38 degrees C 2 days after commencing treatment were significantly more likely to be treatment failures. In resource-poor communities using chloroquine plus chlorpheniramine, the easily identifiable predictors of treatment failure might be used to identify children requiring alternative antimalarial drugs.

Risk factors for gametocyte carriage in uncomplicated falciparum malaria in children.

The risk factors associated with gametocytaemia at presentation and after treatment with different antimalarial drug regimens were evaluated in 767 children enrolled prospectively in 5 antimalarial drug trials between July 1996 and December 2002 in a hyperendemic area of southwestern Nigeria. The children were assigned to one of 6 treatment groups: chloroquine (CQ) only; pyrimethamine-sulfadoxine (PS) only; amodiaquine (AQ) only; CQ combined with chlorpheniramine (CQCP); or PS combined with CQ (CQPS) or AQ (AQPS). At enrolment, 115 (15%) of 767 children were gametocyte carriers. During follow-up, 15.6% of all patients (i.e. 120 patients) developed patent gametocytaemia, which in 85% (102 patients) had developed by day 7 following treatment. In a multiple regression model, 4 factors were found to be independent risk factors for the presence of gametocytaemia at enrolment: male gender (adjusted odds ratio [AOR] = 0.55, 95% confidence interval [CI] 0.36-0.83, P=0.005), absence of fever (AOR = 1.61, 95% CI 1.05-2.5, P=0.03), duration of illness >3 days (AOR=1.57, 95% CI 1.0-2.4, P=0.047), and asexual parasite densities less than 5000/microl (AOR=0.42, 95% CI 0.24-0.73, P=0.002). The presence of patent gametocytaemia at enrolment (AOR=0.04, 95% CI 0.02-0.07, P<0.001) and recrudescence of asexual parasites within 14 days were associated with the presence of gametocytaemia 7 or 14 days after enrolment (AOR=0.5, 95% CI 0.3-0.8, P=0.007). Delay in the time taken to clear the initial parasitaemia (>2 days) was associated with increased risk of subsequent gametocyte carriage. These findings may have implications for malaria control efforts in sub-Saharan Africa where control of the disease depends almost entirely on chemotherapy.

Plasmodium berghei: efficacy and safety of combinations of chloroquine and promethazine in chloroquine resistant infections in gravid mice.

Efficacy and safety of combinations ofChloroquine (CQ) and doses of Promethazine (PR) against CQ resistant Plasmodium berghei infections in gravid mice was evaluated. Parasites were cleared faster in mice treated with CQ combined with doses of PR ranging from 20mg/kg to 50mg/kg (3.4 +/- 0.5 to 2.7 +/- 0.7) compared with CQ alone (4.7 +/- 0.8) (P<0.5). Parturition resulting in live pups in animals treated with CQ and 20mg/ kg and 30mg/kg of PR (81%) was significantly higher than in animals treated with CQ alone (44%) or saline (13%). Mean birth weight of pups delivered by infected gravid animals treated with CQ and 30mg/kg or 40mg/kg of PR (1.51 +/- 0.16 or 1.56 +/- 0.16) was significantly higher than animals treated with CQ alone (1.33 +/- 0.13) (P=0.00004, 0.0014 respectively). No gross malformations were observed in pups delivered by infected or non-infected animals treated with the combinations of chloroquine and Promethazine.

Open randomized study of pyrimethamine-sulphadoxine vs. pyrimethamine-sulphadoxine plus probenecid for the treatment of uncomplicated Plasmodium falciparum malaria in children.

BACKGROUND: Increasing drug resistance in Plasmodium falciparum has necessitated renewed search for cheap, effective alternatives to commonly available antimalarials, chloroquine and pyrimethamine-sulphadoxine, for the treatment of malaria in Africa. Probenecid, an inhibitor of organic anion transporters and multiresistance-associated proteins, can chemosensitize P. falciparum to pyrimethamine and sulphadoxine in vitro, but the clinical significance is unclear. We assessed the safety, treatment efficacy, and effects on gametocyte carriage of adding probenecid to pyrimethamine-sulphadoxine. METHODS: We evaluated 151 children aged 12 years or younger who had uncomplicated P. falciparum malaria. Patients were randomly assigned pyrimethamine-sulphadoxine (25 mg/kg of the sulphadoxine component) or pyrimethamine-sulphadoxine as above plus probenecid 20-25 mg/kg of bodyweight in two divided doses daily for 3 days. The primary endpoints were parasitological cure rates on days 14 and 28. RESULTS: Both regimens were well tolerated; no child was withdrawn because of drug intolerance. Fever (1.9 +/- 1.1 vs. 2.4 +/- 1.2 days, P = 0.02) and parasite clearance (2.3 +/- 0.9 vs. 2.7 +/- 1.1 days, P = 0.04) were significantly shorter, and the parasitological cure rate on day 14 (96.2%vs. 83.5%, P = 0.02) but not day 28 (79.4%vs. 72.6%, P = 0.4), was significantly higher in children treated with pyrimethamine-sulphadoxine-probenecid than in those treated with pyrimethamine-sulphadoxine. Gametocyte carriage was similar with both treatment regimens. CONCLUSIONS: The combination of pyrimethamine-sulphadoxine, and probenecid, at a relatively moderate dose, improved treatment efficacy but had no effect on gametocyte carriage. The pyrimethamine-sulphadoxine-probenecid combination merits further evaluation as a potential treatment for use in Nigeria.

Cotrimoxazole in the treatment of acute uncomplicated falciparum malaria in nigerian children : a controlled clinical trial.

OBJECTIVES: To evaluate the efficacy of cotrimoxazole in the treatment of Plasmodium falciparum malaria and to compare the efficacy of cotrimoxazole with that of pyrimethamine-sulfadoxine, a second-line antimalarial drug, in an area of high malaria transmission. PATIENTS AND METHODS: Children aged between 10 months and 10 years with clinical and parasitological evidence of P. falciparum malaria were randomised to receive either cotrimoxazole or pyrimethamine-sulfadoxine. 145 children (73 and 72, respectively, in the cotrimoxazole and pyrimethamine-sulfadoxine groups) completed the study per protocol and were evaluated. RESULTS: Pretreatment clinical and parasitological parameters were similar in the two treatment groups. The time to clear fever and other symptoms was similar in the two groups: 1.94 +/- 1.10 days versus 2.20 +/- 0.96 days, p > 0.05. Parasite clearance times were also similar: 2.62 +/- 0.91 days versus 2.94 +/- 1.17 days, respectively, for cotrimoxazole and pyrimethamine-sulfadoxine; p > 0.05. The cure rates on days 14, 21 and 28 were, respectively, 84.9, 75.3 and 74.0% for the cotrimoxazole group and 84.7, 80.5 and 75.0% for the pyrimethamine-sulfadoxine group. Both drugs were well tolerated. CONCLUSIONS: These results indicate that cotrimoxazole has similar efficacy to pyrimethamine-sulfadoxine in the treatment of acute uncomplicated P. falciparum malaria in children resident in an endemic area of Southwest Nigeria.

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria.

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1-12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.

Plasmodium falciparum gametocytaemia in Nigerian children: Peripheral immature gametocytaemia as an indicator of a poor response to chloroquine treatment, and its relationship to molecular determinants of chloroquine resistance.

A group of 161 children who presented with acute, symptomatic, uncomplicated, Plasmodium falciparum malaria in an endemic area of Nigeria was investigated. The aims of the study were to determine the clinical characteristics and responses to oral chloroquine (CQ) therapy of children who were gametocytaemic on presentation and those who were not [including those who were found to have developed peripheral immature gametocytaemias (PIG) when checked 72 h after commencing treatment], and to follow the development of PIG 72-336 h after the start of treatment. The 40 consecutive children who did have peripheral gametocytaemia on presentation and the 40 who did not were similar in their clinical characteristics and responses to oral CQ therapy. Nine of the 40 children who did not initially have gametocytaemias but who subsequently developed PIG (stages I-III) 72-336 h after commencing CQ treatment failed the treatment. In order to evaluate the presence of PIG as an indicator of response to CQ, the smears of blood from 81 children--66 classified as resistant to CQ (60, five and one considered RI, RII, RIII, respectively) and 15 who, though considered to have sensitive responses to CQ, cleared their peripheral parasitaemias > or =72 h after commencing CQ therapy--were examined for PIG. Most (42) of the 66 children with CQ-resistant (CQ-R) infections but none of the 15 with sensitive responses had PIG. Among the 66 children with CQ-R infections, the clinical features of those with PIG were generally similar to those without PIG, although those with PIG were more likely to have hepato-splenomegaly and less likely to have hepatomegaly alone. In the children with CQ-R infections, plasma concentrations of CQ on days 7 and 14 were generally above the level necessary to clear sensitive infections in the study area. The results of molecular analyses of isolates from children with both CQ-R infections and PIG revealed that all 14 checked for mutations in pfcrt had the T76 mutation associated with CQ resistance, and that four of the five also checked for mutations in pfmdr1 had the Y86 mutation associated with CQ resistance. The detection of PIG 72 h after the commencement of CQ treatment may be used as a microscopical indicator of a poor response to CQ in children from this endemic area.