Olakunle O. Kassim

Effects of Root Extracts of Fagara zanthoxyloides on the In Vitro Growth and Stage Distribution of Plasmodium falciparum

ABSTRACT The development of resistance by Plasmodium falciparum to conventional drugs poses a threat to malaria control. There is therefore a need to find new, effective, and affordable remedies for malaria, including those derived from plants. This study demonstrates that crude, reverse-phase high-pressure liquid chromatography (RP-HPLC)-semipurified, and RP-HPLC-purified root extracts of Fagara zanthoxyloides inhibit the growth of P. falciparum in vitro, with 50% inhibitory concentrations (IC50s) of 4.90, 1.00, and 0.13 μg/ml, respectively. Roots of F. zanthoxyloides, known as chewing sticks, are widely used for tooth cleaning in West Africa. Microscopic examination of Giemsa-stained slides showed a virtual absence of schizonts in ring-stage synchronized cultures treated with crude extracts at concentrations of 30 to 60 μg/ml during 36 to 48 h of incubation. These observations suggest that the active constituent in the extract may be cytotoxic for P. falciparum trophozoites, thereby inhibiting their development to the schizont stage. A pure bioreactive fraction was subsequently obtained from the chromatographic separations. When this fraction was mixed with pure fagaronine, the mixture coeluted as a single peak on the analytical RP-HPLC column, suggesting that fagaronine may be the active antimalarial constituent of Fagara root extracts. Additional experiments showed that fagaronine also inhibited P. falciparum growth, with an IC50 of 0.018 μg/ml. The results of this study suggest that the antimalarial activity of fagaronine deserves further investigation.

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Inhibition of in-vitro growth of Plasmodium falciparum by Pseudocedrela kotschyi extract alone and in combination with Fagara zanthoxyloides extract.

Roots of Pseudocedrela kotschyi are commonly used as chewing sticks in West Africa. This study examined the effects of the plant extract on the in-vitro growth of Plasmodium falciparum. Ring-stage synchronised cultures of the malaria parasite were exposed to 30 and 60 microg/ml of P. kotschyi extract for 51 h. Aliquots were taken from the cultures every 3 h for preparation of Giemsa-stained thin films, which were evaluated by light microscopy for degree of parasitaemia and stage distribution of parasite development. The extracts did not show any inhibitory effects on the emergence of trophozoites in treated cultures. However, the results indicate that 80% of inhibition of the parasite transformation into schizont was obtained for both tested concentrations (30 and 60 microg/ml). Experiments with (3)H-hypoxanthine incorporation showed an IC(50) of 16 microg/ml for the Pseudocedrela extract. Pseudocedrela was combined with extract of Fagara zanthoxyloides in various concentrations to determine their interactive effects on the in-vitro cultures. Isobologram analysis of the results indicated a synergistic interaction between the two extracts at low concentrations, while interactions at higher concentrations showed antagonistic effects.

Proteinuria and haematuria as predictors of schistosomiasis in children

Examination of urine samples from 922 children from Epe and surrounding communities in south-western Nigeria indicated a 13% prevalence of Schistosoma haematobium infection. Children, 10-14 years of age, accounted for 65% of the disease prevalence. Approximately 79% of the study population was negative for proteinuria, while 52.6% of children with 30 mg% proteinuria were positive for S. haematobium infection. However, 96% and 100% of all children who, respectively, had 30 mg% and 100 mg% proteinuria, in addition to haematuria, were found to be positive for the schistosome infection. This finding indicates that the use of haematuria and proteinuria as a combined diagnostic index significantly increased the sensitivity and specificity of the individual tests. Bacteriuria was found in 8.5% of infected children, compared with 5.2% of the control group. Streptococcus faecalis and E. coli were the two bacteria isolated from the urine specimens.

Antibiotic resistance profile of staphylococci from clinical sources recovered from infants

Infants, children and the aged are among the groups most vulnerable to microbial infections more so when these microbial agents become resistant to antimicrobials. The antibiotic resistant profile of Staphylococcus aureus and selected coagulase negative staphylococci were determined by standard methods. Of the 178 staphylococcal isolates evaluated, 122 were S. aureus and the rest coagulase negative staphylococci. 68% of S. aureus isolates were resistant to amoxicillin, 69.8% to cloxacillin, 51% to augmentin and 71% to tetracycline. However, only 2.6% of the 116 S aureus isolates tested were resistant to gentamycin making the drug a reliable therapeutic agent in the event of failure of other antimicrobials in treating staphylococcal infections at least in this community. Resistance to the penicillin drugs was mediated by the elaboration of β-lactamase by both pathogenic and non-pathogenic staphylococci. The study shows a high rate of cloxacillin resistance and possibly the existence of methicillin resistance among these strains. 80% of the S aureus strains were multi-resistant with 25% of these resistant to three different antibiotics, 21% to 4 and 6.8% to 6 different drugs. Only 1.2% of these S aureus strains were resistant to 7 different antimicrobials underscoring the need to reduce the high incidence of multi-resistance in this community in the event of an epidemic caused by these strains. The study reveals prevalence of multi-resistance among both pathogenic and non-pathogenic staphylococci in the community. Key words : Staphylococci, Staphylococcus aureus , antibiotics, multi-resistance. African Journal of Biotechnology Vol. 4 (8), pp. 810-822

Class-specific antibodies to Bordetella pertussis, Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis in human breast-milk and maternal-infant sera.

Children under 2 years of age are most susceptible to acute respiratory infections caused by Bordetella pertussis, Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis. We analysed milk samples and sera from mother-infant pairs for specific antibodies that may enhance protection against the bacterial pathogens. The results show that the breast-milk samples contained significant titres of specific IgG and IgA antibodies to the four organisms, although the mean IgG antibody levels were higher in maternal sera than in breast-milk. On the other hand, the mean IgA antibody levels to the four organisms were higher in breast-milk than in both maternal and infant sera. IgM antibodies to these organisms were relatively low or absent in many milk and serum samples. Nevertheless, the significant concentrations of specific IgG and IgA antibodies in milk samples may indicate a protective role for breast-milk against the four infections in early childhood.

Assessment of antimalarial effect of ICL670A on in vitro cultures of Plasmodium falciparum.

We tested in vitro the antimalarial properties of ICL670A, a newly developed iron chelator for the long‐term oral treatment of iron overload. Ring‐stage synchronized cultures of Plasmodium falciparum cultured in human erythrocytes were exposed to different concentrations of ICL670A and the conventional iron chelator, desferrioxamine B (DFO), for 48 h. Malarial growth was measured by incorporation of [3H]‐hypoxanthine. ICL670A at 30 µmol/l had marked antimalarial activity that was observable by 6 h after beginning the exposure of ring‐stage parasites to the agent. Over 48 h of culture, malarial growth was significantly lower with ICL670A than with DFO at concentrations of both 30 µmol/l (P = 0·008) and 60 µmol/l (P = 0·001). At 48 h, growth relative to control was 53% with ICL670A and 83% with DFO at concentrations of 30 µmol/l, and 20% with ICL670A and 26% with DFO at concentrations of 60 µmol/l. Standard 50% inhibitory concentrations (IC50s) were similar for ICL670A and DFO. Precomplexation with iron completely abolished the inhibitory effect of ICL670A, indicating that this new agent, like DFO, probably inhibits parasite growth via deprivation of iron from critical targets within the parasite. Further studies to address the question of the antimalarial potential of ICL670A in combination with classic antimalarials would be of interest.

Preponderance of bacterial isolates in urine of HIV-positive malaria-infected pregnant women with urinary tract infection

INTRODUCTION This study examined HIV and malaria co-infection as a risk factor for urinary tract infections (UTIs) in pregnancy. The study group included 74 pregnant women, 20 to 42 years of age, who attended the antenatal clinic at the Specialist Hospital at Akure, Ondo State, Nigeria. METHODOLOGY Forty-four of the pregnant women were either HIV seropositive with malaria infection (HIV+Mal+) or HIV seropositive without malaria (HIV+Mal-). The remaining thirty pregnant women served as controls and included women HIV seronegative but with malaria (HIV-Mal+) and women HIV seronegative without malaria. UTI was indicated by a bacterial colony count of greater than 10⁵/mL of urine, using cysteine lactose electrolyte deficient medium (CLED) as the primary isolation medium. Bacterial isolates were characterized using convectional bacteriological methods, and antibiotics sensitivity tests were carried out using the disk diffusion method. RESULTS A total of 246 bacterial isolates were recovered from the cultures, with a mean of 3.53 isolates per subject. Women who were HIV+Mal+ had the most diverse group of bacterial isolates and the highest frequency of UTIs. The bacterial isolates from the HIV+Mal+ women also showed the highest degree of antibiotic resistance. CONCLUSIONS While pregnancy and HIV infection may each represent a risk factor for UTI, HIV and malaria co-infection may increase its frequency in pregnancy. The higher frequency of multiple antibiotic resistance observed among the isolates, particularly isolates from HIV+Mal+ subjects, poses a serious public health concern as these strains may aggravate the prognosis of both UTI and HIV infection.

The epidemiology of HIV seropositive malaria infected pregnant women in Akure Metropolis, Southwestern Nigeria

Background: HIV increases the risks of malaria in pregnant women, while maternal human immunodeficiency virus (HIV) viral load also facilitates perinatal transmission to neonates. Malaria and HIV coinfection has been shown to exacerbate adverse pregnancy complications. Our study was designed to determine the HIV prevalence of pregnant women at an antenatal clinic in Akure in southwestern Nigeria, investigate the relationship between dual HIV and malaria infection and HIV viral load and CD4+ T cell counts. The study also estimated the risks of adverse pregnancy outcomes in a selected cohort of 74 pregnant women. Materials and Methods: We evaluated the HIV serostatus of 3,225 pregnant women, who attended the antenatal clinic between August 2012 and April 2013. A cohort of 74 pregnant women was selected for the investigation of the relationship between coinfection of HIV and malaria and HIV viral load and CD4+ cell counts. Their HIV status was determined during three trimesters of pregnancy by both HIV-1/2 strips and confirmatory enzyme-linked immunosorbent assay (ELISA) method. Malaria parasitemia was determined by Giemsa-stained thin and thick blood smears. CD4 cell count was by flow cytometry using the CyFlow Counter (Partec, Germany). Viral load estimated by Amplicor HIV-I monitor assay. Results: We found 3.53% prevalence of HIV serostatus among the 3,225 pregnant women who were screened. Forty-four of the 74 subjects were HIV positive and 30 were HIV negative controls. The results show HIV infection among the pregnant women reduced the CD4 cells from a mean of 750 cells/ml for HIV negative women to a mean of 363 cells/ml for HIV seropositive women. Additionally the presence of malaria more than doubled the HIV viral load from a mean of 7,270 ribonucleic acid (RNA) copies/ml for HIV positive women without malaria to 15,148 RNA copies/ml for HIV positive women with malaria. Conclusion: In this study, HIV infection significantly increased risk of acquiring malaria in pregnant women (odds ratio (OR) = 2.27). Dual HIV/malaria infections exacerbated adverse pregnancy outcomes

Plasmodium falciparum: Activity of artemisinin against Plasmodium falciparum cultured in sickle trait hemoglobin AS and normal hemoglobin AA red blood cells

The presence of sickle hemoglobin causes accumulation of hemoglobin degradative products that favor oxidative reaction in erythrocytes. Artemisinin derivatives exert antiparasite effects through oxidative reactions within infected erythrocytes. Using [(3)H]-hypoxanthine incorporation, we therefore did an in vitro comparison of IC(50) values for artemisinin in Plasmodium falciparum-infected erythrocytes from sickle cell trait (AS) and normal (AA) individuals. IC(50) values for chloroquine served as control. Without drugs, parasite growth was similar in AA and AS erythrocytes. Gender, age and blood group of donors had no significant effects on parasite growth. IC(50) value for artemisinin was 27+/-14nM in AS (N=22) compared to 24+/-9nM (N=27) in AA erythrocytes (P=0.4). IC(50) values for chloroquine were also similar in AA (22+/-8nM) and AS (20+/-11nM) erythrocytes. These results show no evidence of elevated artemisinin activity on P. falciparum in AS erythrocytes in vitro.