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.

In vitro cytotoxicity studies of 20 plants used in Nigerian antimalarial ethnomedicine.

Twenty plants identified and selected from Southwest and Middle belt Nigerian antimalarial ethnopharmacology were evaluated for in vitro cytotoxicity using the brine shrimp lethality assay. The methanol extracts of 20 plant samples from 11 plant families were subjected to the assay. Of the studied plants, Lippia multiflora and Morinda lucida bark were found to be cytotoxic, with LC(50) values of 1.1 and 2.6 microg/ml, respectively. The least toxic plant extract was Bridelia micrantha (LC(50) value >9.0 x 10(6) microg/ml). Most of the plants were found to be relatively non-toxic.

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Efficacy of herbal remedies used by herbalists in Oyo State Nigeria for treatment of Plasmodium falciparum infections--a survey and an observation.

In the course of evaluating the contribution of phytomedicine to possible drug discovery of antimalarial drugs, an ethnomedical survey of specialized children traditional clinics was done. In the observational multi center study, efficacy of eight different herbal remedies, each consisting of 3-8 ingredients and administered by herbalists were investigated in clients enrolled in the six traditional clinics in Oyo (urban center) and Otu (rural center) of Oyo State, Nigeria. The clients, aged between six months and fifteen years with clinical symptoms of malaria were enrolled in the clinics of the herbalists, as their usual practice. Oral informed consents were obtained from their parents or guardians. Microscopic diagnosis of malaria infection was used to evaluate parasitaemia and validate efficacy of herbal remedies. Results of the analysis showed that, of the 163 clients of the herbalists, only 62 (30 from Oyo, 32 from Otu) had microscopically confirmed P. falciparum infection. Only results from 54 clients (29/30 (Oyo) and 25/32 (Otu) with P. falciparum infection could be evaluated. Plasmodium falciparum infection in 88% (23/29) of clients from Oyo responded to treatment with the herbal remedies while cure rate in clients from Otu was 42% (13/25). Parasite densities ranged from 171 to 53,613 parasites/microl blood and 87 to 36,209 parasites/microl blood in patients from Oyo and Otu respectively. The herbalists administered the remedies and Gossypium arboreum, Anarcadium occidentalis, Citrus medica, Phyllanthus amarus and Lippia multiflora were the main ingredients in the efficacious remedies. The herbalists gave detailed descriptions of each of the 8 herbal remedies proffered. The results confirm the efficacy of two of the eight herbal remedies, thereby validating the role of ethnomedicine as a possible source for the discovery of new chemotherapeutic agents in the treatment of P. falciparum malaria.

Malaria diagnosis: false negative parasight-F tests in falciparum malaria patients in Nigeria.

This study was designed to assess the relative reliability of microscopy, the dipstick technique based on the detection of Plasmodium falciparum-specific histidine rich protein II (HRPII) (Parasight-F) and PCR assays in diagnosing falciparum malaria infections in Nigerian children. The prevalence of P. falciparum infections in enrolled patients was 100% by microscopy. Parasite density ranged from 329 to 81,194 parasites/microL of blood, with a geometric mean parasite density of 5168 parasites/microL of blood. The sensitivity of the HRPII based dipstick, PCR and microscopy were 80%, 92% and 100% respectively. A false negative rate of 20% was observed with Parasight-F as compared with microscopy. The parasitemia in patients with false negative Parasight-F tests ranged from 319 to 54,680 parasites/microL of blood. Detailed PCR analysis of Isolates obtained from five out of the eight patients who exhibited a negative Parasight-F test, showed that the average numbers of P. falciparum clones in these five isolates were: 1.7 +/- 1.02 with MSPI, 3.2 +/- 1.3 with MSP2 and 1.4 +/- 1.72 with GLURP. Comparison of microscopy and HRPII results showed a significant (p=0.009) difference as opposed to microscopy and PCR (p=0.239). This study showed that caution should be exercised when excluding P. falciparum infections on the basis of HRPII based dipstick results alone. Microscopy or PCR diagnosis where possible, should be carried out in order to confirm negative P. falciparum HRPII-based dipstick tests.

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.

Molecular analysis of Plasmodium falciparum recrudescent malaria infections in children treated with chloroquine in Nigeria.

Parasite genotyping by a polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in 50 of 160 Nigerian children taking part in a chloroquine efficacy study in Ibadan, Nigeria. A finger prick blood sample was taken from each child before and after treatment to identify recrudescent parasites. By investigating allelic variation in three polymorphic antigen loci, merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP), we determined parasite diversity in the population and in the infected host. DNA from pretreatment and post-treatment samples from 47 of the 50 patients who failed therapy was successfully amplified by the PCR. The MSP-1, MSP-2, and GLURP genotypes in all samples showed extensive diversity, indicating polyclonal infections. The average number of clones per infection in pre-treatment sample was 2.5 with MSP-1, 4.9 with MSP-2, and 2 with GLURP. The extent of multiplicity decreased significantly (P = 0.016) in posttreatment samples. Multiplicity of infection and initial parasite density were not age dependent. Comparison of the variant alleles in pretreatment and post-treatment samples of each patient indicates that 26 of the 47 children had genuinely recrudescent disease. Conversely, post-treatment samples from five children showed completely new genotypes, indicating either a previously sequestered population of parasites or a newly acquired infection. Overall, this study has shown the diversity and complexity of P. falciparum population in Ibadan, Nigeria. The study has also shown the dynamics of P. falciparum infections in this population before and after chloroquine treatment in an area of high malaria transmission.

Human urinary schistosomiasis transmission foci and period in an endemic town of Ijebu North, Southwest Nigeria.

Human urinary schistosomiasis transmission foci and period were investigated in an endemic town in Ijebu North, Southwest Nigeria. Three important streams (Areru, Eri-Oru and Ojupon) were sampled using long-handled scoop and a pair of tongs, between September 2002 and October 2003. Out of the 3500 freshwater snails collected, six species were identified which are Lanistes libycus (46.7%), Potadoma moerchii (31.4%), Melanoides tuberculata (1.5%), Bulinus globosus (18.2%), B.forskalii (0.2%) and Afrogyrus coretus (2.1%). 611 (96.1%) of the B.globosus recorded were from Eri-Oru and Areru streams 588 (96.2%) of the B.globosus from these two streams were recorded during little or no rainfall period when the snail infection rate range was 2.3 to 21.0%. Artificial cercarial infection of laboratory mice yielded terminally- spined eggs characteristic of Schistosoma haematobium. 16.2% and 5.0% of A. coretus collected from Eri-Oru in January and March 2003 respectively, shed cercariae which were not furcocercous. The results are discussed in relation to the nutritional significances of L. libycus and P.moerchii, findings from preliminary study and rainfall pattern in the study area. The study showed that human urinary schistosomiasis transmission occurred mainly in the dry season and that Eri-Oru and Areru streams were the main transmission foci.

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.

Cultural categorization of febrile illnesses in correlation with herbal remedies used for treatment in Southwestern Nigeria.

The ethnographic study was conducted in two communities in Oyo State in Southwestern Nigeria. The study sites consisted of a rural and an urban local government area located in the tropical rain forest zone of Nigeria. The study was designed to obtain information on febrile illnesses and herbal remedies for treatment with the aim of identifying potential antimalarial drugs. The study revealed that fever is a general term for describing illnesses associated with elevated body temperature. The indigenous Yoruba ethnic population has categorized fever based on symptoms and causes. The present communication is the result of focus group discussion and semi-structured questionnaire administered to traditional healers, herb sellers, elders and mothers. This was on types of fevers, symptoms and causes of febrile illnesses. The investigation also included use of traditional herbs in the prevention and treatment of the illnesses in the two communities.A total of 514 respondents were interviewed. This was made up of 266 (51.8%) from Atiba local government area (LGA), an urban centre while 248 (48.2%) respondents were interviewed from Itesiwaju LGA, a rural community. The LGAs are located in Oyo State of Nigeria. The respondents proffered 12 types of febrile illnesses in a multiple response answering system in Yoruba language. The most common ones (direct translation into English) were: yellow fever (39.1%), typhoid (34.8%), ordinary (28.8%), rainy season (20.8%) and headache (10.5%) fevers, respectively. Perceived causes of each of the febrile illnesses included stress, mosquito bites, unclean water, rains and over exposure to the sun. Methods of fever prevention were mainly with the use of herbal decoctions, powdered herbs, orthodox medications and maintenance of proper hygiene. Of a total of 112 different herbal remedies used in the treatment of the febrile illnesses compiled from the study, 25 recipes are presented. Recipes consisted of 2-7 ingredients. Oral decoctions (84%), oral powders (63%), use as soaps and creams (40%) in a multiple response system, were the most prevalent routes of administration of prepared herbs used in the treatment of the fevers. Boiling in water or alcohol was the most common method used in the preparation of the remedies. The four most frequently mentioned (multiple response system) plants in the Southwest ethnobotany for fevers were Azadirachta indica (87.5%), Mangifera indica (75.0%), Morinda lucida (68.8%) and Citrus medica (68.8%).

Loa loa and Mansonella perstans infections in Ijebu north, western Nigeria: a parasitological study.

The prevalence and intensity of Loa loa and Mansonella perstans were studied in three villages of the Ijebu north area in Ogun State, western Nigeria. Blood samples were collected by finger-pricking from 373 (181 males, 192 females) subjects with an age range of 4 to 55 years. The blood samples were examined microscopically for the presence of microfilariae (mf). L. loa and M. perstans were present in the blood samples in 39 (10.5%) and 12 (3.2%) of the subjects, respectively. Neither of the infections were found to be sex-dependent. The geometric mean of the L. loa intensity was 1.8 mf per 50 microl of blood, while that of M. perstans was 1.5 mf per 50 microl. The prevalence of mixed infections of both L. loa and M. perstans was 1.0%.

HIV seropositivity in Nigerians with lymphoproliferative malignancies.

One hundred and four Nigerians with lymphoproliferative malignancies were tested for antibodies to human immunodeficiency virus types 1 and 2 (HIV-1 and 2). All the patients with high grade lymphomas in this series were seronegative for HIV-1 and 2. Only 1 of the 104 patients had antibody to HIV-1 and none had antibody to HIV-2. Although the single positive result would suggest a higher HIV-1 seropositivity rate among the patients compared with the general Nigerian population, it should be interpreted with caution. This preliminary data emphasizes the need for an urgent countrywide study of the problem among this category of patients in Nigeria in view of the low prevalence rate of HIV-1 and 2 in the country.

PIP: Several workers have reported an association between lymphoproliferative malignancies and HIV infection. The revised diagnostic criteria for AIDS which include HIV-seropositivity in patients with Non-Hodgkin's lymphoma of the small, noncleaved, immunoblastic and large cell types are consistent with the observed relationship between HIV infection and lymphoproliferative malignancies. Following this revised definition, it was deemed necessary to determine the prevalence of HIV infection among the patients with lymphoproliferative malignancies seen in the University College Hospital, Ibadan, Nigeria. 104 newly-diagnosed Nigerian patients with lymphoproliferative malignancies seen at the hospital between December 1986 to May 1990 were tested for antibodies to HIV-1 and HIV-2. One patient was HIV-1-seropositive and none were HIV-2-seropositive. All patients with high-grade lymphomas were seronegative for both viruses. These preliminary data indicate the urgent need for a countrywide study designed to learn more about the relationship between HIV infection and lymphoid malignancies in Nigeria. The authors stress that while Burkitt's lymphoma coexisting with HIV-seropositivity is currently taken as diagnostic of AIDS, it is important to establish that the viral infection was acquired before the tumor developed in each patient. This observation is important due to the commonality of Burkitt's lymphoma in Nigeria where some hospitals are still unable to screen blood for HIV before transfusion.

Malaria and loaisis among blood donors at Ibadan, Nigeria.

115 blood donors were screened for the presence of malaria and other blood parasites at the Blood Bank, University College Hospital, Ibadan, between January and July, 1984. 11.3% of these had blood parasites; 7.8% had Plasmodium falciparum with parasitaemias from 0.03-0.2%, and 3.5% had Loa loa microfilaraemia. No other blood parasites were observed. Serological examination by the indirect fluorescent antibody test revealed that 86% of the donors had malaria antibody with reciprocal titres ranging from 16 to 512. There was no significant association between ABO blood group and malaria parasitaemia or antibody titre.