In Vitro effects of petroleum ether, dichloromethane, methanolic and aqueous leaf extracts of Eucalyptus grandis on selected multidrug-resistant bacteria.

BACKGROUND: The emergence and spread of antimicrobial resistance is of grave concern, requiring the search for newer and more effective antimicrobials to combat infections caused by resistant microbes. This study assessed the antimicrobial effects of Eucalyptus grandis crude extracts against selected multidrug resistant bacteria. METHODOLOGY: Four different crude leaf extracts of E. grandis were prepared using petroleum ether, dichloromethane, methanol, and water, with the aid of the Soxhlet extraction method. These were screened against methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant Pseudomonas aeruginosa, and multidrug resistant Escherichia coli, using the agar well diffusion method. Phytochemical screening was carried out to evaluate the bioactive phytochemical constituents responsible for the antimicrobial effect. RESULTS: Each of the extracts, except for the one prepared from water, had antimicrobial activity against the screened bacteria. The non-polar petroleum ether extract had the highest antimicrobial activity (19.33-24.33 mm), including bactericidal effects, compared to the medium polar dichloromethane and polar methanol extracts, which recorded zone diameter ranges of 14.33-16.67 mm and 16.33-17.67 mm, respectively. The Gram-negative bacteria (E. coli and P. aeruginosa) were the least susceptible in comparison with the Gram-positive bacterium (MRSA), probably owing to differences in their cell wall structures. Furthermore, phytochemical screening indicated the presence of alkaloids, tannins, saponins, terpenoids, and flavonoids. CONCLUSION: The findings suggest that E. grandis could be potentially useful in the treatment of infections caused by multidrug resistant bacteria.

The Impact of Plasmodium Infection on Placental Histomorphology: A Stereological Preliminary Study.

Background: Malaria during pregnancy may threaten the mother's health and cause serious structural damage to the internal architecture of the placenta, which subsequently affects the pregnancy outcome. A better understanding of the impact of malaria parasites on the placenta morphology is crucial for better management of pregnant women and their babies. Aim: To assess by stereology the histomorphology of selected placental structures in placenta malaria compared with normal placentae at term. Method: A total of 10 placentae comprising 5 controls and 5 cases were selected from 50 placentae that were collected at term (38 weeks ± 2 weeks) from the maternal delivery suit of Korle-Bu Teaching Hospital in Accra, Ghana. Blood from the placentae was collected for both rapid diagnostic test and microscopic examinations. Samples collected were examined for Plasmodium parasites, after which they were classified as study group (Plasmodium positive) or control (Plasmodium negative). Stereological quantification using systematic uniform random sampling technique with test point and intersection counting of photomicrographs were employed to estimate the mean volume densities of syncytial knots, syncytial necrosis, foetal capillaries, and intervillous spaces of the placentae on a total of 1,600 photomicrographs. Results: Out of the fifty placental samples from the maternal side tested for Plasmodium, six representing 12% were found to be infected with the parasite by both rapid diagnostic test and microscopy. On stereological assessment, the mean volume density of syncytial knots was significantly higher in the placental malaria group compared with the control placentae at term (P = 0.0080), but foetal capillaries (P = 0.7813), intervillous spaces (P = 0.8078), and syncytial necrosis (P = 0.8249) were not significantly different. Conclusion: This preliminary result indicates that placental malaria may cause significant increase in the syncytial knots but not foetal capillaries, intervillous spaces, or syncytial necrosis. This finding signifies early maturation of the placenta and may be crucial in understanding perinatal outcomes.

In Vitro Assessment of Antiplasmodial Activity and Cytotoxicity of Polyalthia longifolia Leaf Extracts on Plasmodium falciparum Strain NF54.

BACKGROUND: Malaria is one of the most important life-threatening infectious diseases in the tropics. In spite of the effectiveness of artemisinin-based combination therapy, reports on reduced sensitivity of the parasite to artemisinin in Cambodia and Thailand warrants screening for new potential antimalarial drugs for future use. Ghanaian herbalists claim that Polyalthia longifolia has antimalarial activity. Therefore, antiplasmodial activity, cytotoxic effects, and antioxidant and phytochemical properties of P. longifolia leaf extract were investigated in this study. METHODOLOGY/PRINCIPAL FINDINGS: Aqueous, 70% hydroethanolic and ethyl acetate leaf extracts were prepared using standard procedures. Antiplasmodial activity was assessed in vitro by using chloroquine-sensitive malaria parasite strain NF54. The SYBR® Green and tetrazolium-based calorimetric assays were used to measure parasite growth inhibition and cytotoxicity, respectively, after extract treatment. Total antioxidant activity was evaluated using a free radical scavenging assay. Results obtained showed that extracts protected red blood cells against Plasmodium falciparum mediated damage. Fifty percent inhibitory concentration (IC50) values were 24.0±1.08 µg/ml, 22.5±0.12 µg/ml, and 9.5±0.69 µg/ml for aqueous, hydroethanolic, and ethyl acetate extracts, respectively. Flavonoids, tannins, and saponins were present in the hydroethanolic extract, whereas only the latter was observed in the aqueous extract. Aqueous and hydroethanolic extracts showed stronger antioxidant activities compared to the ethyl acetate extract. CONCLUSIONS/SIGNIFICANCE: The extracts of P. longifolia have antiplasmodial properties and low toxicities to human red blood cells. The extracts could be developed as useful alternatives to antimalarial drugs. These results support claims of the herbalists that decoctions of P. longifolia are useful antimalarial agents.

Risk of heavy metal ingestion from the consumption of two commercially valuable species of fish from the fresh and coastal waters of Ghana.

BACKGROUND: The need to evaluate the human health safety of fishery resources remain urgent in the mist of the ever-increasing fear of heavy metal toxicity from the consumption of Ghana's fisheries resource, as a consequence of pollution from several anthropogenic activities including artisanal gold mining. Nevertheless, the bigeye grunt (Brachydeuterus auritus) and Bagrid catfish (Chrysichthys nigrodigitatus) remain commercially valuable fish species in West Africa and continue to attract high patronage. METHOD: Forty-five specimens each of C. nigrodigitatus and B. auritus collected from the Weija Dam and the Tema Fishing Habour in Ghana, between June and September 2016, were analysed for seven heavy metals using Atomic Absorption Spectrometry. RESULT: Lead and Cadmium were below detection in all samples while Cu was not detected in B. auritus. Levels of the remaining metals (mg kg-1) were below FAO/WHO maximum permissible limits in fish and occurred in the rank order Se (3.5) > Zn (2.34) > Cu (0.59) > As (0.37) > Hg (0.19) in C. nigrodigitatus and Se (2.97) > Zn (2.28) > Hg (0.31) > As (0.21) in B. auritus. Only As in C. nigrodigitatus recorded Estimated Weekly Intake (EWI) greater than FAO/WHO Provisional Tolerable Weekly Intake (PTWI). Also, As in C. nigrodigitatus and Hg in B. auritus had Targeted Hazard Quotient (THQ) greater than 1 for individuals consuming the fishes on daily basis and therefore, raising concerns. However, for both species of fish, cancer risk of As was 1 in 10,000,000,000 and modified Health Benefits values of Se (HBVSe) were positive indicating the health risks that might accompany Hg exposure would be negated. Since toxicity depends on the concentration and quantity of a pollutant consumed, safe maximum consumption rate of C. nigrodigitatus based on As concentrations was 0.21 mg per day and that of B. auritus was 0.058 mg per day for Hg. With an average of 0.227 kg fish per meal of an adult human, these translated into not more than 24 C. nigrodigitatus and nine (9) B. auritus meals in a month but because fish is consumed at 0.0685 kg per person per day in Ghana, these values respectively translates to 93 and 30 safe days of consumption per month. CONCLUSION: At the rate of 0.0685 kg fish per person per day that fish is consumed in Ghana, the consumption of the two species of fish in Ghana would essentially be of little or no consequence to consumers.

Twelve-month longitudinal parasitological assessment of lymphatic filariasis-positive individuals: impact of a biannual treatment with ivermectin and albendazole.

OBJECTIVE: Mass drug administration (MDA) for the control of lymphatic filariasis (LF), in Ghana, started in the year 2000. While this had great success in many implementation units, there remain areas with persistent transmission, after more than 10 years of treatment. A closer examination of the parasite populations could help understand the reasons for persistent infections and formulate appropriate strategies to control LF in these areas of persistent transmission. MATERIALS AND METHODS: In a longitudinal study, we assessed the prevalence of microfilaraemia (mf) in two communities with 12 years of MDA in Ghana. In baseline surveys 6 months after the National MDA in 2014, 370 consenting individuals were tested for antigenaemia using immunochromatographic test (ICT) cards and had their mf count determined through night blood surveys. 48 ICT positives, of whom, 17 were positive for mf, were treated with 400 µg/kg ivermectin + 400 mg albendazole and subsequently followed for parasitological assessment at 3-month intervals for 1 year. This overlapped with the National MDA in 2015. RESULTS: There was a 68% parasite clearance 3 months after treatment. The pre-treatment mf count differed significantly from the post-treatment mf counts at 3 months (P = 0.0023), 6 months (P = 0.0051), 9 months (P = 0.0113) and 12 months (P = 0.0008). CONCLUSION: In these settings with persistent LF transmission, twice-yearly treatment may help accelerate LF elimination. Further large-scale evaluations are required to ascertain these findings.

Activity of Herbal Medicines on Plasmodium falciparum Gametocytes: Implications for Malaria Transmission in Ghana.

BACKGROUND: Malaria still remains a major health issue in Ghana despite the introduction of Artemisinin-based combination therapy (ACT) coupled with other preventative measures such as the use of insecticide treated nets (ITNs). The global quest for eradication of malaria has heightened the interest of identifying drugs that target the sexual stage of the parasite, referred to as transmission-blocking drugs. This study aimed at assessing the efficacy and gametocydal effects of some commonly used herbal malaria products in Ghana. METHODOLOGY/PRINCIPAL FINDINGS: After identifying herbal anti-malarial products frequently purchased on the Ghanaian market, ten of them were selected and lyophilized. In vitro drug sensitivity testing of different concentrations of the herbal products was carried out on asexual and in vitro generated gametocytes of the 3D7 strain of Plasmodium falciparum. The efficacies of the products were assessed by microscopy. Cultures containing low dose of RT also produced the least number of late stage gametocytes. Two of the herbal products CM and RT inhibited the growth of late stage gametocytes by > 80% at 100 µg/ml whilst KG was the most inhibitory to early stage gametocytes at that same concentration. However at 1 µg/ml, only YF significantly inhibited the survival of late stage gametocytes although at that same concentration YF barely inhibited the survival of early stage gametocytes. CONCLUSIONS/SIGNIFICANCE: Herbal product RT (Aloe schweinfurthii, Khaya senegalensis, Piliostigma thonningii and Cassia siamea) demonstrated properties of a highly efficacious gametocydal product. Low dose of herbal product RT exhibited the highest gametocydal activity and at 100 µg/ml, RT exhibited >80% inhibition of late stage gametocytes. However inhibition of asexual stage parasite by RT was not optimal. Improving the asexual inhibition of RT could convert RT into an ideal antimalarial herbal product. We also found that generally C. sanguinolenta containing herbal products exhibited gametocydal activity in addition to high asexual efficacy. Herbal products with high gametocydal activity can help in the fight to reduce malaria transmission.

Transmission indices and microfilariae prevalence in human population prior to mass drug administration with ivermectin and albendazole in the Gomoa District of Ghana.

BACKGROUND: The Lymphatic Filariasis Elimination Programme in Ghana involves annual mass drug administration (MDA) of ivermectin and albendazole to persons living in endemic areas. This is repeated annually for 4-6 years to span across the reproductive lifespan of adult worms. In order to stimulate participation of community members in the MDA programme, this study was carried out to understand local views on transmission, management and prevention of the disease. The study also presents baseline transmission indices and microfilariae prevalence in the human population in eight endemic communities of coastal Ghana prior to the MDA. METHODS: A descriptive survey was carried out to explore perceptions on causes, treatment and prevention of lymphatic filariasis. Perceptions on community participation in disease control programmes were also assessed. After participants were selected by cluster sampling and 100 µl of blood sampled from each individual and examined for mf microfilariae. A similar volume of blood was used to determine the presence of circulating filarial antigen. Mosquitoes were collected simultaneously at all sites by human landing catches for 4 days per month over a six-month period. All Anopheles mosquitoes were dissected and examined for the larval stages of the parasite following which molecular identification of both vector and parasite was done. RESULTS: Eight hundred and four persons were interviewed, of which 284 (32.9%; CI 31.1-34.5) acknowledged elephantiasis and hydrocoele as health related issues in the communities. Thirty-three people (3.8%; CI 2.1-5.5) thought sleeping under bed net could help prevent elephantiasis. Microfilariae prevalence was 4.6% (43/941) whiles 8.7% (75/861) were positive for circulating filarial antigen. A total of 17,784 mosquitoes were collected, majority (55.8%) of which were Anopheles followed by Culex species (40%). Monthly biting rates ranged between 311 and 6116 bites/person for all the eight communities together. Annual transmission potential values for An. gambiae s.s. and An. funestus were 311.35 and 153.50 respectively. CONCLUSION: Even though the highest mf density among inhabitants was recorded in a community that had the lowest Anopheles density with Culex species constituting 95% of all mosquitoes collected, Anopheles gambiae s.s. and An. funestus remained the main vectors.

Implications of low-density microfilariae carriers in Anopheles transmission areas: molecular forms of Anopheles gambiae and Anopheles funestus populations in perspective.

BACKGROUND: Previous studies have shown a general reduction in annual transmission potential (ATP) of Anopheles species after mass drug administration (MDA) in lymphatic filariasis endemic communities. Whereas results obtained from a monitoring programme after three years of MDA revealed a decrease in ATP of Anopheles funestus this was not the same for An. gambiae s.s. in Ghana. In this study, the ability of these vectors in transmitting Wuchereria bancrofti in nine lymphatic filariasis endemic communities in Gomoa District of Ghana after four rounds of MDA with ivermectin and albendazole was investigated. METHODS: After mass screening of inhabitants in these communities, twelve consenting volunteers with different intensities of microfilariae (mf) slept under partly opened mosquito nets as sources of mf blood meal. Hourly collection of mosquitoes and finger-pricked blood were taken from 21.00 to 06.00 hours the following day. For each hour, half of the mosquitoes collected were immediately killed and dissected for mf. The remaining half were maintained up to 13 days for parasite maturation. Parasitaemia and infection rates in the mosquitoes were determined by microscopy. The mosquitoes were identified by microscopy and molecular techniques. RESULTS: A total of 1,083 participants were screened and the overall parasite prevalence was 1.6% with mf intensities ranging from 0 to 59 per 100 µl and geometric mean intensity of 1.1 mf per ml of blood. Of the 564 mosquitoes collected, 350 (62.1%) were Anopheles spp., from which 310 (88.6%) were An. funestus and 32 (9.1%) An. gambiae. Six anopheline mosquitoes (1.7%) were found infected with L1, but no larva was observed in any of the mosquitoes maintained up to 13 days. Molecular studies showed all An. gambiae s.l. to be An. gambiae s.s., of which 21 (70%) were of the M molecular form. CONCLUSION: At low-level parasitaemia after 4 rounds of MDA, there was no recovery of infective stage larvae of W. bancrofti in An. funestus s.l. as well as M and S forms of An. gambiae.

Basophil depletion downregulates Schistosoma mansoni egg-induced granuloma formation.

Granuloma formation around parasite eggs during schistosomal infection is considered to be controlled by Th2 cytokines. However, it is still controversial which cell populations are responsible for the host Th2 cytokine-dependent granuloma formation. Basophils have recently attracted attention because of their ability to produce large amounts of IL-4. Therefore, we investigated whether basophils play an essential role in the induction of granuloma formation induced by Schistosoma mansoni eggs. Together with our previous observation that basophil numbers increased markedly in the spleen at 7 weeks postinfection, immunohistochemical staining using anti-mMCP8 monoclonal antibody (mAb) showed basophil infiltration in the granulomatous lesions formed around parasite eggs. To examine the roles of basophils more directly, we treated mice with anti-CD200R3 mAb to deplete basophils. Depletion of basophils resulted in a reduction of basophil number with concomitant downregulation of egg granuloma formation at 7 weeks postinfection. Moreover, we observed a significant reduction in the size of egg granulomas formed in basophil-depleted mice in the pulmonary granuloma model. Taken together, these findings indicated that basophils are essential for S. mansoni egg-induced granuloma formation, and this may serve as a novel therapeutic target in ameliorating the pathology of schistosomiasis.

Interleukin-4 (IL-4) and IL-13 suppress excessive neutrophil infiltration and hepatocyte damage during acute murine schistosomiasis japonica.

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.

Administrative practices of health professionals and use of artesunate-amodiaquine by community members for treating uncomplicated malaria in southern Ghana: implications for artemisinin-based combination therapy deployment.

OBJECTIVE: To investigate the use of artemisinin-based combination and monotherapy by community members and the administrative practices of health professionals in treating malaria in Ghana. METHOD: This study is a community-based cross-sectional survey in 11 rural and urban areas in southern Ghana. Using the interviewer method, close-ended questionnaires were administered to community members. Similar questionnaires were also administered in health facilities, community pharmacies and licensed chemical shops. RESULTS: A total of 1085 individuals comprising 959 non-health professionals and 126 health professionals were interviewed. Fifty-seven per cent of the community members visit pharmacies/drug stores as the first point of call when they suspect malaria. According to the participating drug sellers, artemether-lumefantrine (AL) is the most prescribed/sold anti-malarial drug (59.2%), followed by dihydroartemisinin (35%), sulfadoxine-pyrimethamine (33.0%) and artesunate-amodiaquine (AS-AQ) (27.2%). The majority of customers who visit pharmacies or drug stores without prescription have their anti-malarial drug selected by the shop attendant; in situations like that, dihydroartemisinin and artesunate monotherapies are sold just as AS-AQ and AL. Chloroquine is still sold by some drug vendors, 5 years after its proscription. CONCLUSION: Whereas the use of AS-AQ and AL are acceptable, the frequent use of dihydroartemisinin and artesunate monotherapy threatens the future of ACTs.

Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro.

BACKGROUND: In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes. METHODS: The prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test. RESULTS: All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00). CONCLUSION: The pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.

Plasmodium falciparum: differing effects of non-esterified fatty acids and phospholipids on intraerythrocytic growth in serum-free medium.

Different combinations of non-esterified fatty acids (NEFA) had variable effects on intraerythrocytic growth of Plasmodium falciparum. All stages of the parasite cultured in medium supplemented with cis-9-octadecenoic acid (C18:1-cis-9), hexadecanoic acid (C16:0), phospholipids (Pld) and bovine albumin free of NEFA were similar to those grown in complete growth medium. Three typical growth patterns indicating suppressed schizogony (SS), suppressed formation of merozoites (SMF), and inhibited invasion of merozoites (IMI) resulted from culture in other combinations of lipids. Unsaturated or saturated NEFA with longer or shorter carbon chains than C18:1-cis-9 or C16:0, higher degree of unsaturation, and trans-forms mainly resulted in SS and SMF effects. However, IMI or partial IMI was observed with tetradecanoic acid or octadecanoic acid enriched with C18:1-cis-9, and cis-9-hexadecenoic acid plus C16:0. Isoforms of C18:1-cis-9 also mainly resulted in partial IMI. SMF also occurred with C18:1-cis-9 plus C16:0 in the absence of Pld. Thus different NEFA exerted distinct roles in erythrocytic growth of the parasite by sustaining development at different stages.