Spectrophotometric studies of a novel Gedunin-2-Hydroxypropyl- ß-cyclodextrin binary system.

BACKGROUND: Gedunin, a limonoid, is linked with antimalarial, anticancer and anti-allergic activities. This study was aimed at preparing an inclusion complex of gedunin and 2-hydroxypropyl-p-cyclodextrin (HBD) to increase solubility of-gedunin in polar solvents which will increase absorption and bioavailability in vivo and thus enhance pharmacological effects. MATERIALS AND METHODS: Gedunin was obtained from the hexane extract of Entandrophragma angolense heartwood by column and preparative thin layer chromatography. The structure was previously confirmed by spectroscopic means (NMR). The electronic absorption spectra data of the complexes formed between gedunin. and HBD in various solvents was determined using the UV-VIS spectrophotometer. The stoichiometry of inclusion was determined by Job's method of continuous variation. RESULTS: Evidence of interaction was observed between gedunin and HBD in the various solvents but gedunin and its complex with HBD exhibited sharp absorption bands in acetate buffer (pH 3.5).The spectrophotometric titrations showed curves with a single point of inflexion when the experiment was carried'out at 25°C (298 K) and 37°C (310 K). A stoichiometric ratio of 1:1 for complex formation was obtained. The formation constants (K,) obtained at 25°C and 37°C.were 9.539 x.10³ M⁻¹ and .1.853 x 104 M⁻¹ respectively. Thermodynamic considerations revealed hydrophobic interaction between gedunin and HBD. CONCLUSION: A stable inclusion complex of gedunin and HBD was formed at room and body temperature. This complex formation involved trapping of poorly soluble gedunin into the hydrophobic core of the cyclodextrin and may enhance the pharmacological activity of gedunin in vivo.

Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan, Southwest Nigeria.

The emergence of insecticide resistance in Anopheles (Diptera: Culicidae) mosquitoes has great implications for malaria control in Nigeria. This study aimed to determine the dynamics of insecticide susceptibility levels and the frequency of knock-down resistance (kdr) mutations (L1014F) in wild Anopheles coluzzii Coetzee & Wilkerson sp. n. and Anopheles gambiae Giles from the Ojoo and Bodija areas of Ibadan, in southwest Nigeria. Insecticide susceptibility to pyrethroids, organophosphates, carbamates and organochlorines was assessed using World Health Organization (WHO) bioassays. A subset of the mosquitoes exposed to pyrethroids and DDT was used for species and molecular form identification; kdr genotyping was determined using the TaqMan real-time polymerase chain reaction assay. The mosquitoes were resistant to pyrethroids and DDT but completely susceptible to organophosphates and carbamates. Bodija samples (n = 186) consisted of An. gambiae (91.4%) and An. coluzzii (8.1%) and included one An. coluzzii/An. gambiae hybrid specimen. All mosquitoes screened in Ojoo (n = 26) were An. gambiae. The 1014F kdr mutation was detected at frequencies of 24.5 and 5.8% in Bodija and Ojoo, respectively. No correlation was observed between kdr genotypes and resistance phenotypes. The results indicate that metabolic resistance probably plays an important role in the development of resistance and highlight the need to implement insecticide resistance management strategies.

Evaluation of the comparative efficacy and safety of artemether-lumefantrine, artesunate-amodiaquine and artesunate-amodiaquine-chlorpheniramine (Artemoclo™) for the treatment of acute uncomplicated malaria in Nigerian children.

OBJECTIVE: To evaluate the comparative efficacy and safety of artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ) and artesunate-amodiaquine-chlorpheniramine (AQC) for the treatment of acute uncomplicated malaria among Southwest Nigerian children. SUBJECTS AND METHODS: One hundred and sixty children aged 6 months to 14 years with acute uncomplicated malaria were randomized to AL (n = 53), ASAQ (n = 53), or AQC (n = 54). Enrollees were seen daily on days 0-3 and then on days 7, 14, 21, 28 and 42 for clinical and parasitological evaluations. Paired samples of genomic DNA at enrolment and at the time of recurrent parasitaemia were genotyped using nested PCR to distinguish between reinfection and recrudescence. Detailed haematological and biochemical evaluations were carried out in a subset of enrollees on days 0, 7 and 28 as part of a safety evaluation. RESULTS: Of the 160 children, 144 (90%) completed the study. The mean fever clearance times and parasite clearance times for AL, ASAQ and AQC were comparable (p = 0.94 and p = 0.122, respectively). On day 14, the adequate clinical and parasitological response (ACPR) for AL and AQC was 100% and for ASAQ it was 90% (p = 0.39). The PCR-uncorrected results on days 28 and 42 and the ACPR-corrected results on day 42 were similar for all drugs (p = 0.62 and p = 0.56, respectively). AQC resulted in the best parasite clearance and haematological recovery on day 2 (p = 0.022 and p = 0.018, respectively). Biochemical parameters were not adversely affected by the three artemisinin-based combination therapies (ACTs) and these were well tolerated. CONCLUSION: The three ACTs were efficacious and safe, but AQC resulted in a better haematological recovery on day 2 and higher cure rates throughout the study period.

AN INVESTIGATION INTO THE ANTIMALARIAL EFFECT OF METHANOLIC EXTRACT OF PAULLINIA PINNATA LEAVES IN PLASMODIUM BERGHEI INFECTED MICE AND COURSE OF INFECTION.

AIM: The aim of this study was to investigate the antimalarial activity of methanolic leaves extract of Paullinia pinnata on chloroquine-sensitive Plasmodium berghei NK 65 infected mice. METHODOLOGY: The curative study was conducted in thirty-six Wistar albino mice of both sexes which were divided into six groups of six animals each. The animals were infected with P. berghei NK 65. Group I was the negative control and received the vehicle (10% DMSO). Group II received no treatment. Groups III and IV were the positive controls and received chloroquine (CQ) (10mg/kg) and artesunate (4 mg/kg)-amodiaquine (10mg/kg) combination (ACT) respectively. Groups V and VI received 100mg/kg and 200mg/kg doses of the extract respectively. Administration was done orally once for three or four days for the standard drugs or the extract/vehicle respectively. The percentage parasitaemia, packed cell volume (PCV), body weight and death was monitored on days 0, 1, 2, 3, 4 and 11 (7 day post administration). The study of the course of infection of P. berghei was monitored in eighteen Wistar albino mice of both sexes which were similarly grouped, infected and treated for 3 days. Group A received the vehicle (distilled water) only. Group B was treated with CQ (10 mg/kg) and Group C with ACT. The percentage parasitaemia and death was monitored from day 0 to day 30 (27 day post administration). RESULTS: In the curative study, the extract suppressed parasitaemia at both doses on day 4. The group treated with 200mg/kg dose showed a higher percentage chemosuppression though not significant. The course of infection study revealed that recrudescence occurred on day 8 in the CQ treated group which lasted until day 23 after which the recrudescence was lost without re-treatment. A similar result was observed in the ACT group. CONCLUSION: The methanolic leaves extract of Paullinia pinnata has weak anti-malarial property. Chloroquine-sensitive P. berghei NK65 loses credibility and needs to be revalidated biannually.

Anti-inflammatory, Anti-nociceptive and Total polyphenolic Content of Hydroethanolic Extract of Ocimum gratissimum L. Leaves.

Ocimum gratissimum has been reported in several ethnopharmacological surveys as a plant readily accessible to the communities and widely used with a lot of therapeutic potentials. In this study, we aimed to experimentally evaluate the anti-inflammatory effects of hydro-ethanolic extract in animal models of inflammation and nociception and membrane stabilization assay. O gratissimum leaves hydroethanolic extract was subjected to phytochemical screening and spectrophotometric quantification of polyphenolics. The extract was investigated for anti-inflammatory effects in carrageenan -induced paw oedema and cotton pellet - induced granuloma in rats. The antinociceptive effects were investigated in acetic acid -induced writhing in mice and formalin test in rats. Animals were randomly divided into groups; negative control, extract treated (200 -800 mg/kg) and indomethacin (10 mg/kg) standard reference groups. In- vitro anti-inflammatory activity was performed by testing for membrane stability in heat/hypotonic solution -induced rat erythrocytes destabilization assay. Phytochemical screening revealed presence of saponins, tannins, alkaloids, flavonoids, terpenoids, and cardenolides. Quantification of the polyphenolic content revealed the presence of appreciable quantities of phenolics and flavonoids. Carrageenan-induced paw oedema, cotton-pellet granuloma, acetic acid -induced writhing and formalin induced paw licking tests showed that hydroethanolic extract of O gratissimum possess anti-inflammatory and anti-nociceptive effects. The extract did not induce gastric lesion formation in stomach of cotton-pellet granuloma rats. The extract was more efficient at reducing membrane destabilization than indomethacin in the membrane stability assay. These results suggest that hydroethanolic extract of O gratissimum leaves exhibits anti-inflammatory and anti-nociceptive effects in the animals.

The protective effects of methyl jasmonate against adriamycin--induced hepatic and renal toxicities.

The aim of the study was to investigate the protective effect of methyl jasmonate (MJ) in adriamycin (ADR) induced hepatic and renal toxicities. 36 BALB/c mice were randomly divided into control, ADR (20 mg/kg), MJ (50 mg/kg) only, MJ (100 mg/kg) only, MJ (50 mg/ kg) + ADR, MJ (100 mg/kg) + ADR groups (n = 6). The 2 doses of MJ was administered for 7 days in MJ only groups, ADR was administered intraperitoneally on the 8th day after pretreatment with the 2 different doses of MJ while ADR was administered on the 8th day only for the ADR only group. The malondialdehyde (MDA), glutathione (GSH), H2O2 generation, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine in the liver, kidneys and serum samples as applicable were estimated. Tissue MDA, H2O2 generation, and GST activity were markedly elevated while GSH content, CAT and SOD activities were significantly reduced in the tissues when compared to the control (p < 0.05). Pretreatment with MJ ameliorated ADR toxicities, with a significant reduction in serum urea concentration, ALT activity, MDA level, H2O2 generation, GST activity and a significant elevation in GSH content, CAT and SOD activities in the organ tissues. MJ induced significant reduction in MDA level and increase of GSH content in liver and kidney tissues. This study suggests that MJ may play an overall protective effect on ADR-induced toxicities in liver and kidneys and the inhibition of tissue peroxidative damage might contribute to this beneficial effect.

Effects of low protein diet and pregnancy on course of Plasmodium berghei infection in mice.

Pregnancy and malnutrition influence the severity or trend of malaria especially in sub-Saharan Africa where parasitic infections are highly predominant. This study was used to evaluate the combined effects of low protein diet and pregnancy on the course of Plasmodium berghei infection in mice. Thirty female BALB/c mice were divided into six groups viz: Non-infected mice fed on normal diet (NIND), Infected mice fed on normal diet (IND), Noninfected mice fed on low protein diet (NILP), Infected mice fed on low protein diet (ILP), Non-infected gravid mice fed on low protein diet (NIGLP) and Gravid infected mice fed on low protein diet (GILP). Malaria parasite count, packed cell volume, body weight and plasma nitric oxide (NO) production were determined. Data were compared statistically across the groups using Student t-test and ANOVA. Parasite detection in peripheral blood was delayed in ILP (day 7) and GILP (day 11) relative to IND (day 3). The peak parasitaemia and mean survival time were significantly lower (p < 0.05) in GILP relative to other infected groups. GILP could not carry the pregnancy to term. Nitric oxide production was observed to increase more rapidly in IND relative to ILP after parasite detection with a peak production by day 15. Mortality commenced in both groups afterwards. Low protein diet delayed the peak production of NO supporting its protective influence on malaria infection. However, the combined effects of low protein diet and pregnancy resulted in early mortality and inability of mice to carry pregnancy to term.

Effect of antimalarial drugs and malaria infection on oxidative stress in pregnant women.

This work studied the effect of malaria infection and antimalarial drugs on oxidative stress in 259 pregnant and nonpregnant women at Ade-Oyo hospital, Ibadan, Nigeria. Oxidative stress was determined by measuring serum lipid peroxidation, ascorbic acid, and reduced glutathione (GSH) levels using spectrophotometer. The results showed that mean lipid peroxidation was significantly higher (p < 0.05) in malaria positive than malaria negative women, while GSH and ascorbic acid levels were significantly (p < 0.05) reduced. The parasite density was significantly reduced in patients who had taken antimalarial drugs relative to those without. While mean ascorbic acid and GSH levels were significantly reduced in those who had taken drugs as compared with those without drugs, the lipid peroxidation level was significantly higher in them. The increase in lipid peroxidation and decrease in GSH and ascorbic acid levels in women who were malaria positive and in those who had taken drugs is indicative of oxidative stress.

Blood banking in a malaria-endemic area: evaluating the problem posed by malarial parasitaemias.

The emergence and wide dissemination of drug-resistant malarial parasites underscore the need to prevent post-transfusion malaria. In Nigeria, as in most of sub-Saharan Africa, however, blood donors are not routinely screened for malarial infection. Recently, 391 consecutive potential blood donors in a malaria-endemic area of south-western Nigeria were each checked for malarial parasitaemia using three methods: microscopy (all samples), OptiMAL (315 samples) and/or the Clinotech Malaria Cassette (142 samples). OptiMAL detects parasite-specific lactate dehydrogenase whereas the Clinotech test detects the surface proteins of merozoites and sporozoites. Microscopy revealed parasitaemias in 79 (20.2%) of the potential donors, the levels of parasitaemia varying from 34 to 6289 asexual parasites/microl (mean=445/microl). The prevalence of malarial parasitaemia, as detected by microscopy, was significantly higher during the rainy season than in the dry season (27.3% v. 5.5%; P<0.0001). There was no significant association between patent parasitaemia and fever (i.e. an axillary temperature > or =37.5 degrees C), blood group, gender or anaemia. The corresponding prevalences of malarial parasitaemia detected using the rapid diagnostic tests were 3.8% (12/315) for OptiMAL and 57.8% (82/142) for the Clinotech. With the results of the microscopy used as the 'gold standard', OptiMAL gave a sensitivity of only 16.0% but a specificity of 98.5%. The corresponding values for the Clinotech tests were 69.2% and 50.0%, respectively. It would clearly be beneficial to include screening for malaria parasitaemia in the routine investigation of potential blood donors in Nigeria, especially during the rainy season, when the risk of transfusion-transmitted malaria appears relatively high.

Potential toxicity of chlorpheniramine plus chloroquine for the treatment of childhood malaria.

OBJECTIVES: To compare the adverse effects of two regimens of chlorpheniramine plus chloroquine (CP+CQ) in children who live in a country where chloroquine resistant malaria is endemic. METHODS: 99 children with acute uncomplicated malaria were randomised into two treatment groups. Group I received high dose chlorpheniramine (6 mg +12 mg/day for 7 days in children = 5 years; 8 mg + 18 mg/day for 7 days in those >5 years) plus chloroquine 10 mg/kg daily for 3 days. Group II received a 50% higher dose of chlorpheniramine plus chloroquine 10 mg/kg daily for 3 days. Outcome measures were vital signs, clinical response and parasite clearance on days 0-7 and day 14. RESULTS: Parasite clearance, fever clearance and cure rate were comparable for the two groups. Drowsiness occurred in 66.7% of high dose and 86.3% of higher dose CP+CQ subjects (p = 0.05). Compared to children treated with high dose, those treated with higher dose CP+CQ had significantly lower respiratory rates on day 2 (p = 0.001), day 6 (p = 0.015), and on day 14 (p = 0.003). CONCLUSION: The higher rates of drowsiness and lower respiratory rates in children treated with higher dose CP+CQ calls for caution in the clinical application of the higher dose combination. The higher dose has no additional benefit and may in fact be dangerous.

High efficacy of two artemisinin-based combinations (artemether-lumefantrine and artesunate plus amodiaquine) for acute uncomplicated malaria in Ibadan, Nigeria.

OBJECTIVE: To test the hypothesis that artesunate plus amodiaquine (ASAQ) is as effective as artemether-lumefantrine (AL) in the treatment of acute uncomplicated malaria in Nigerian children. METHODS: In an open label, randomized controlled clinical trial, children aged 6 months to 10 years were randomized to receive artesunate (4 mg/kg daily) plus amodiaquine (10 mg/kg daily) or AL (5-14 kg, one tablet; 15-24 kg, two tablets and 25-34 kg, three tablets twice daily). Both drug regimens were given for 3 days and follow-up was for 28 days. RESULTS: A total of 132 children (66 in each group) were randomized to receive either ASAQ or AL. Day 28 cure rates in the per protocol (PP) population were 93% for ASAQ and 95% for AL (OR = 0.71, 95% CI = 0.12-3.99, rho = 0.66). Using Kaplan-Meier product-limit estimates of failure, the median survival time for ASAQ was 21 days and for AL 28 days (P = 0.294). PCR corrected day 28 cure rate for PP populations were 98.4% for ASAQ and 100% for AL. Both drugs were well-tolerated. CONCLUSION: ASAQ is as effective as AL and both combinations were efficacious and safe.

Anti-protozoan activities of Harungana madagascariensis stem bark extract on trichomonads and malaria.

AIM OF THE STUDY: The ethanolic stem bark extract of Harungana madagascariensis (Hypericaceae), (Choisy) Poir were evaluated for their activities on Trichomonas gallinae (Rivolta) Stabler isolated from the pigeon (Columba livia). It was also tested for their anti-malarial activity on N67 Plasmodium yoelii nigeriensis (in vivo) in mice and on Plasmodium falciparum isolates in vitro. MATERIALS AND METHODS: The anti-trichomonal screening was performed in vitro using Trichomonas gallinae culture. The minimum lethal concentration (MLC) is the lowest concentration of the test extract in which no motile organisms were observed. The anti-malarial effects were determined in-vivo for suppressive, curative and prophylactic activities in mice receiving a standard inoculum size of 1 x 10(7) (0.2 ml) infected erythrocytes of Plasmodium yoelii nigeriensis intraperitoneally, and the in vitro was performed against 3 isolates of Plasmodium falciparum in a candle jar procedures. RESULTS: The IC(50) of the extract and metronidazole (MDZ) (Flagyl) on Trichomonas gallinae at 48 h are 187 and 1.56 microg/ml. The IC(50) of the extract, chloroquine (CQ) and artemether (ART) on Plasmodium falciparum are between 0.052 and 0.517 microg/ml for the extract and 0.021 and 0.0412 microg/ml for ART and CQ, respectively. The actions of the extract in in vivo study on Plasmodium yoelii nigeriensis showed that in both suppressive and prophylactic tests the percentages chemo-suppressive were between 28.6-44.8% and 30.2-78.2% respectively, while only 80 mg/kg of the extract reduced the parasitaemia level when compared to the control and the standard drugs in curative test. CONCLUSIONS: Harungana madagascariensis stem bark extract therefore exhibited significant anti-protozoan effects against Trichomonas and Plasmodium both in vivo and in vitro.

Prevalence of malaria at booking among antenatal clients in a secondary health care facility in Ibadan, Nigeria.

The prevalence of malaria parasitemia at booking was studied in 1,848 pregnant women in a secondary hospital in Ibadan, Nigeria. Main outcome variables were patent parasitemia and fever. 8.4% hadpatent malaria parasitaemia. Most clients (89%) with parasitemia were asymptomatic. Febrile subjects booked at an earlier gestational age [22.7 versus 24.2 weeks] than afebrile patients (p = 0.0052). Anemia was more prevalent among patients with patent parasitemia than those without (58.1% versus 22.6%, p < 0.0001). Malaria parasitaemia was higher among nulliparous women than other parity groups (p < 0.0001). Symptomatic malaria was associated with early booking for antenatal care and malaria parasitemia was a significant determinant of anemia. The prevalence of malaria parasitaemia in this study is much lower than in previous reports.

In vitro antimalarial activity of methylene blue against field isolates of Plasmodium falciparum from children in Southwest Nigeria.

BACKGROUND & OBJECTIVES: Methylene blue (MB), a thiazine dye is used in the treatment of various methemoglobinaemias. However, sporadic reports have shown some antimalarial therapeutic effect when administered to patients with clinical manifestations of malaria. The inhibitory concentration of schizont maturation and antimalarial activity of MB have not been fully elucidated. The present study therefore aimed at determining the antimalarial activity of MB in Plasmodium falciparum isolates obtained from children with malaria using standard in vitro drug susceptibility test. METHODS: Twenty children (8 boys and 12 girls) within the age range 4.5-11.5 yr were enrolled into the study and 2 ml of blood withdrawn aseptically. The standard microtest technique of schizont inhibition assay was used to culture fresh isolates obtained from P. falciparum infected patients. Chloroquine (CQ) and quinine (QN) were used as reference standards for in vitro drug susceptibility tests. RESULTS: The mean 50 per cent inhibitory concentration (IC(50)) values were 9.59 +/- 3.25nM, 196 +/-21.11nM and 607 +/- 27.41nM for MB, CQ and QN respectively. Ten of the 14 isolates were sensitiveto MB, 11 were sensitive to CQ while nine were sensitive to QN. Three isolates were resistant to CQ,and of these, two were sensitive to MB and one was sensitive to QN. INTERPRETATION & CONCLUSION: This preliminary study showed that MB has high antimalarial activity comparable with CQ and QN and may be used as a potent schizonticidal drug against CQ-resistant isolates.

The disposition of chloroquine and its main metabolite desethylchloroquine in volunteers with and without chloroquine-induced pruritus: evidence for decreased chloroquine metabolism in volunteers with pruritus.

The pharmacokinetics of chloroquine and its main metabolite desethylchloroquine have been carried out in volunteers with and without chloroquine-induced pruritus. It was shown that the volunteers with pruritus tended to metabolize chloroquine slower than the volunteers without pruritus because the metabolic ratio was lower in the volunteers with pruritus than that in the volunteers without pruritus. However, the overall pharmacokinetic patterns were comparable between the two groups and agreed with published data. The 24-hour urinary collections in the two groups of volunteers indicated that the volunteers with pruritus excreted more chloroquine (although not statistically significant) than the volunteers without pruritus. This also indicates that they metabolized less chloroquine. There were no side effects of note in any of the volunteers. The volunteers who gave positive histories of chloroquine-induced pruritus had mild episodes of itching after intake of the drug; the pruritus subsided within 48 hours in all instances.

Lack of association between falciparum malaria parasitemia and acute diarrhea in Nigerian children.

It is widely believed that malaria causes diarrhea. Yet, national and international diarrheal diseases control programs are silent about the overlap between these two major public health problems that coexist in most tropical countries. To test the hypothesis that malaria is associated with diarrhea and to define the role of malaria in morbidity due to diarrhea, 522 children 6-60 months of age presenting with acute diarrhea to the Children's Emergency Ward of the University College Hospital in Ibadan, Nigeria were routinely screened by means of thin and thick blood films for malaria parasitemia. Controls, without diarrhea, were studied in parallel. Detailed clinical features were recorded for every patient. Sixty-eight (13%) of the 522 diarrhea patients screened had malaria parasitemia. Among the controls (who had similar distributions of admission temperature, hemoglobin types, glucose-6-phosphate dehydrogenase deficiency, and prior treatment with antimalarial drugs), parasitemia was not significantly different, occurring in 56 (17.9%) of 313. In the dry season, however, a significantly higher prevalence of parasitemia was observed among the control group (15.5%) than in the diarrhea group (7.0%) (P = 0.004). Parasitemia was significantly more common in the dehydrated diarrhea patients than their well-hydrated counterparts (25% of 56 versus 11% of 466; P < 0.005). There were no significant differences in admission temperature, the presence of vomiting, or the home use of oral rehydration fluids between the dehydrated and the well-hydrated subsets of diarrhea patients. Consideration of parasite densities did not alter any of the foregoing relationships. These data contradict the widely held view that diarrhea is a symptom of malaria or that malaria causes diarrhea. They do, however, provide support for examining blood smears at least in dehydrated children with diarrhea in malaria-endemic areas and giving immediate antimalarial therapy to those who have malaria parasitemia.

High rate of mixed and subpatent malarial infections in southwest Nigeria.

The rate of malarial parasitemia in children and adults was assessed by microscopy and the polymerase chain reaction in a holoendemic area in Nigeria. A high rate of subpatent Plasmodium falciparum parasitemia (19.6%) was found. Plasmodium malariae and P. ovale infections were common in a rural area (26.1% and 14.8%) but were observed sporadically in individuals from an urban area. Simultaneous infections with P. falciparum, P. malariae, and P. ovale were frequent in the rural area (11.7% triple infections). The rate of triple infections was higher than expected from the prevalences of each species (P < 0.00001). Spleen enlargement was associated with mixed infections of P. falciparum and P. malariae (odds ratio [OR] = 5.9, 95% confidence interval [CI] 3.0-11.7) and less frequently observed in individuals without detectable parasitemia (OR = 0.06, 95% CI = 0.01-0.3). Spleen enlargement and titers of antibodies to schizonts were positively correlated with parasite densities. The results also suggest that in some individuals a long-lasting subpatent parasitemia might occur.

Short report: high prevalence and imbalanced age distribution of the Plasmodium falciparum dihydrofolate reductase gene Asn108 mutation in an area of low pyrimethamine usage in Nigeria.

Resistance of Plasmodium falciparum to pyrimethamine is associated with a non-silent point mutation of the parasite dihydrofolate reductase (DHFR) gene (Ser108 --> Asn108). Wide-scale use of antimalarials is thought to contribute to the emergence of drug resistance. In 131 P. falciparum-infected children in rural Nigeria, the frequency of the resistant Asn108 genotype was assessed by enzymatic restriction digestion of polymerase chain reaction-amplified DHFR sequences and compared with residual pyrimethamine blood levels. The prevalence of the Asn108 variant was 41.2%. In 18.3% of the isolates, both the Asn108 and the wild-type alleles were present. In contrast to the high prevalence of resistant genotypes, residual pyrimethamine blood levels were detected in only 4%. Furthermore, age was found to be a determinant of the parasite genotype since the proportion of Asn108 variants decreased with age (P < 0.05). These findings indicate that additional, unidentified factors, rather than selection by residual drug levels alone, might be responsible for the emergence of pyrimethamine-resistant parasite genotypes.

Impact of subpatent multi-species and multi-clonal plasmodial infections on anaemia in children from Nigeria.

Childhood anaemia in sub-Saharan Africa is often caused by Plasmodium falciparum malaria. The influence of subpatent, multi-species and polyclonal infections with malaria parasites on haematological parameters was assessed in 1996/97 in clinically healthy children in Nigeria. Of the 228 children studied, 64% were anaemic by the WHO age-dependent criteria. A univariate analysis of risk factors indicated that the prevalence of anaemia was dependent on the number of Plasmodium species detected by species-specific PCR (P < 0.0001). Furthermore, the prevalence of anaemia increased gradually with the complexity (P < 0.003) as well as with the extent of P. falciparum parasitaemia (P < 0.0001). A logistic regression analysis revealed that individuals with an enlarged spleen tended to be anaemic. The number of Plasmodium species by which an individual was infected was independently associated with anaemia (P < 0.03). ANOVA revealed that the age-corrected values for haemoglobin (Hb) and red blood cells (RBCs) were mainly influenced by the occurrence of mixed infections. Haematological parameters were also influenced by the number of different P. falciparum clones by which an individual was infected. Hb levels and RBC counts were further diminished by additional infections with P. malariae and/or P. ovale. However, the effect of multi-species infections on haematological parameters exceeded that of multi-clonal infections.

Molecular epidemiology and activity of erythrocyte G6PD variants in a homogeneous Nigerian population.

OBJECTIVE: To determine accurately the relative frequencies and enzyme activities of the polymorphic variants of G6PD in a homogeneous population in Nigeria. SETTING: Abanla village in the outskirt of Ibadan city and the University College Hospital, Ibadan Nigeria. SUBJECT: Seven hundred and twenty one subjects who belong to the Yoruba tribe of Southwestern Nigeria. METHOD: Two mls of blood was withdrawn from each subject. G6PD activity was quantified by spectrophotometry. DNA was extracted for genotyping of G6PD by PCR. RESULTS: G6PD deficiency was 23.9% and 4.6% in males and females respectively. The gene frequencies of the different G6PD variants (Gd) were in accordance with expected Hardy-Weinberg equilibrium. Only GdA-1 type was found in subjects with deficient variants. G6PD activity decreased significantly with age among non-deficient individuals. The range of enzyme activities was wide and overlapping among the different G6PD variants. CONCLUSION: G6PD deficiency was very high in the population. The gene frequencies were similar to previous findings. Molecular methods of typing G6PD allowed for direct and accurate genotyping of the enzyme in both males and females without having to combine several methods.