In vitro effects and mechanisms of action of Bidens pilosa in Trypanosoma brucei.

Background and aim: African trypanosomiasis poses serious health and economic concerns to humans and livestock in several sub-Saharan African countries. The aim of the present study was to identify the antitrypanosomal compounds from B. pilosa (whole plant) through a bioactivity-guided isolation and investigate the in vitro effects and mechanisms of action against Trypanosoma brucei (T. brucei). Experimental procedure: Crude extracts and fractions were prepared from air-dried pulverized plant material of B. pilosa using the modified Kupchan method of solvent partitioning. The antitrypanosomal activities of the fractions were determined through cell viability analysis. Effects of fractions on cell death and cell cycle of T. brucei were determined using flow cytometry, while fluorescence microscopy was used to investigate alterations in cell morphology and distribution. Results and conclusion: The solvent partitioning dichloromethane (BPFD) and methanol (BPFM) fractions of B. pilosa exhibited significant activities against T. brucei with respective half-maximal inhibitory concentrations (IC50s) of 3.29 µg/ml and 5.86 µg/ml and resulted in the formation of clumpy subpopulation of T. brucei cells. Butyl (compound 1) and propyl (compound 2) esters of tryptophan were identified as the major antitrypanosomal compounds of B. pilosa. Compounds 1 and 2 exhibited significant antitrypanosomal effects with respective IC50 values of 0.66 and 1.46 µg/ml. At the IC50 values, both compounds significantly inhibited the cell cycle of T. brucei at the G0-G1 phase while causing an increase in G2-M phase. The results suggest that tryptophan esters may possess useful chemotherapeutic properties for the control of African trypanosomiasis.

Screening for tick-borne and tick-associated viruses in ticks collected in Ghana.

Ticks are blood-sucking arthropods that transmit many pathogens, including arboviruses. Arboviruses transmitted by ticks are generally referred to as tick-borne viruses (TBVs). TBVs are known to cause diseases in humans, pets, and livestock. There is, however, very limited information on the occurrence and distribution of TBVs in sub-Saharan Africa. This study was designed to determine the presence and distribution of ticks infesting dogs and cattle in Ghana, as well as to identify the tick-borne or tick-associated viruses they harbour. A more diverse population of ticks was found to infest cattle (three genera) relative to those infesting dogs (one genus). Six phleboviruses and an orthonairovirus were detected in tick pools screened by RT-PCR. Subsequent sequence analysis revealed two distinct phleboviruses and the previously reported Odaw virus in ticks collected from dogs and a virus (16GH-T27) most closely related to four unclassified phleboviruses in ticks collected from cattle. The virus 16GH-T27 was considered a strain of Balambala tick virus (BTV) and named BTV strain 16GH-T27. Next-generation sequencing analysis of the BTV-positive tick pool detected only the L and S segments. Phylogenetic analysis revealed that BTV clustered with viruses previously defined as M-segment-deficient phleboviruses. The orthonairovirus detected in ticks collected from cattle was confirmed to be the medically important Dugbe virus. Furthermore, we discuss the importance of understanding the presence and distribution of ticks and TBVs in disease prevention and mitigation and the implications for public health. Our findings contribute to the knowledge pool on TBVs and tick-associated viruses.

Determining vector competence of Aedes aegypti from Ghana in transmitting dengue virus serotypes 1 and 2.

BACKGROUND: Dengue virus (DENV) is a mosquito-borne arbovirus transmitted by Aedes mosquitoes, but is not endemic in all areas where this vector is found. For example, the relatively sparse distribution of cases in West Africa is generally attributed to the refractory nature of West African Aedes aegypti (Ae. aegypti) to DENV infection, and particularly the forest-dwelling Ae. aegypti formosus. However, recent studies have shown these mosquitoes to be competent vectors within some West African countries that have suffered outbreaks in the past, such as Senegal. There is however little information on the vector competence of the Ae. aegypti in West African countries such as Ghana with no reported outbreaks. METHODS: This study examined the vector competence of 4 Ae. aegypti colonies from urban, semi-urban, and two rural locations in Ghana in transmitting DENV serotypes 1 and 2, using a single colony from Vietnam as control. Midgut infection and virus dissemination were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while the presence and concentration of DENV in the saliva of infectious mosquitoes was determined by the focus forming assay. RESULTS: There were significant differences in the colonies' susceptibility to virus infection, dissemination, and transmission. All examined Ghanaian mosquitoes were refractory to infection by DENV serotype 2, while some colonies exhibited potential to transmit DENV serotype 1. None of the tested colonies were as competent as the control group colony. CONCLUSIONS: These findings give insight into the possible risk of outbreaks, particularly in the urban areas in the south of Ghana, and highlight the need for continuous surveillance to determine the transmission status and outbreak risk. This study also highlights the need to prevent importation of different DENV strains and potential invasion of new highly vector-competent Ae. aegypti strains, particularly around the ports of entry.

Towards understanding antimicrobial activity, cytotoxicity and the mode of action of dichapetalins A and M using in silico and in vitro studies.

Dichapetalum madagascariense Poir (Dichapetalaceae) is traditionally used to treat bacterial infections, jaundice, urethritis and viral hepatitis in Africa. Its root contains a broad spectrum of biologically active dichapetalins. To evaluate the plant's effect on human MCF-7 cells and its' antibacterial and antiparasitic potentials, we isolated and identified the known dichapetalins A and M from the roots. Both dichapetalins were tested on six bacterial strains (Shigella flexneri, Bacillus cereus, Salmonella paratyphi B, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus) and two parasite strains; Trypanosoma brucei brucei, and Leishmania donovani using the Alamar Blue assay system. Dichapetalins A and M were more potent against B. cereus with IC50 values of 11.15 and 3.15 µg/ml, respectively, compared to the positive control ampicillin (IC50 = 19.50 µg/ml). Dichapetalins A (IC50 = 74.22 µg/ml) and M (IC50 = 72.34 µg/ml) were less active against T. b. brucei, compared to the standard Suramin (IC50 = 4.96 µg/ml). Dichapetalin M showed moderate activity against L. donovani (Amphotericin B: IC50 = 0.21 µg/ml) with an IC50 of 16.80 µg/ml. In human MCF-7 cells expressing the NR1I2 receptor, the activity of dichapetalin M was higher (IC50 = 4.71 µM and 3.95 µM) for 48 and 72 h of treatment, respectively compared to Curcumin with IC50 of 17.49 µM and 12.53 µM for 48 and 72 h of treatment, respectively. Results from in vitro expression studies with qPCR confirmed an antagonistic effect of dichapetalin M on PXR (NR1I2) signaling; supporting the PXR signaling pathway as a possible mode of action of dichapetalin M as predicted by in silico results. These findings confirm previous studies that D. madagascariense can be a source of potential lead compounds for development of novel antibiotic, antiparasitic and anticancer medicines, and provide further insights into the mechanism of action of the dichapetalins.

Isolation and Antitrypanosomal Characterization of Furoquinoline and Oxylipin from Zanthoxylum zanthoxyloides.

In the absence of vaccines, there is a need for alternative sources of effective chemotherapy for African trypanosomiasis (AT). The increasing rate of resistance and toxicity of commercially available antitrypanosomal drugs also necessitates an investigation into the mode of action of new antitrypanosomals for AT. In this study, furoquinoline 4, 7, 8-trimethoxyfuro (2, 3-b) quinoline (compound 1) and oxylipin 9-oxo-10, 12-octadecadienoic acid (compound 2) were isolated from the plant species Zanthoxylum zanthoxyloides (Lam) Zepern and Timler (root), and their in vitro efficacy and mechanisms of action investigated in Trypanosomabrucei (T. brucei), the species responsible for AT. Both compounds resulted in a selectively significant growth inhibition of T. brucei (compound 1, half-maximal effective concentration EC50 = 1.7 µM, selectivity indices SI = 74.9; compound 2, EC50 = 1.2 µM, SI = 107.3). With regards to effect on the cell cycle phases of T. brucei, only compound 1 significantly arrested the second growth-mitotic (G2-M) phase progression even though G2-M and DNA replication (S) phase arrest resulted in the overall reduction of T. brucei cells in G0-G1 for both compounds. Moreover, both compounds resulted in the aggregation and distortion of the elongated slender morphology of T. brucei. Analysis of antioxidant potential revealed that at their minimum and maximum concentrations, the compounds exhibited significant oxidative activities in T. brucei (compound 1, 22.7 µM Trolox equivalent (TE), 221.2 µM TE; compound 2, 15.0 µM TE, 297.7 µM TE). Analysis of growth kinetics also showed that compound 1 exhibited a relatively consistent growth inhibition of T. brucei at different concentrations as compared to compound 2. The results suggest that compounds 1 and 2 are promising antitrypanosomals with the potential for further development into novel AT chemotherapy.

Entomological Assessment of the Status and Risk of Mosquito-borne Arboviral Transmission in Ghana.

Entomological surveillance is one of the tools used in monitoring and controlling vector-borne diseases. However, the use of entomological surveillance for arboviral infection vector control is often dependent on finding infected individuals. Although this method may suffice in highly endemic areas, it is not as effective in controlling the spread of diseases in low endemic and non-endemic areas. In this study, we examined the efficiency of using entomological markers to assess the status and risk of arbovirus infection in Ghana, which is considered a non-endemic country, by combining mosquito surveillance with virus isolation and detection. This study reports the presence of cryptic species of mosquitoes in Ghana, demonstrating the need to combine morphological identification and molecular techniques in mosquito surveillance. Furthermore, although no medically important viruses were detected, the importance of insect-specific viruses in understanding virus evolution and arbovirus transmission is discussed. This study reports the first mutualistic relationship between dengue virus and the double-stranded RNA Aedes aegypti totivirus. Finally, this study discusses the complexity of the virome of Aedes and Culex mosquitoes and its implication for arbovirus transmission.

Antitrypanosomal Effects of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler Extracts on African Trypanosomes.

African trypanosomiasis is a disease caused by the parasitic protozoa of the Trypanosoma genus. Despite several efforts at chemotherapeutic interventions, the disease poses serious health and economic concerns to humans and livestock of many sub-Saharan African countries. Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Z. zanthoxyloides LZT) is a plant species of important phytochemical and pharmacological relevance in the subtropical zones of the African continent. However, the mechanisms of its antitrypanosomal effects in African trypanosomes remain to be elucidated. The aim of the study was to determine the in vitro effects and mechanisms of action of Z. zanthoxyloides LZT (root) fractions against Trypanosoma brucei. T. brucei (GUTat 3.1 strain), L. donovani (D10 strain), P. falciparum (3D 7 strain), Jurkat cells, and Chang liver cells were cultivated in vitro to the log phase in their respective media at 37°C. Crude extracts and fractions were prepared from air-dried pulverized plant material of Z. zanthoxyloides LZT (root) using the modified Kupchan method of solvent partitioning. Half-maximal inhibitory concentrations (IC50) were determined through the alamar blue cell viability assay. Effects of fractions on cell death and cell cycle of T. brucei were determined using flow cytometry. Fluorescence microscopy was used to investigate the effects of fractions on the morphology and distribution of T. brucei. Antitrypanosomal compounds of fractions were characterized using high-performance liquid chromatography (HPLC) and attenuated total reflectance infrared (ATR-IR) spectroscopy. Methanol, butanol, and dichloromethane fractions were selectively active against T. brucei with respective IC50 values of 3.89, 4.02, and 5.70 µg/ml. Moreover, methanol, butanol, and dichloromethane fractions significantly induced apoptosis-like cell death with remarkable alteration in the cell cycle of T. brucei. Furthermore, dichloromethane and methanol fractions altered the morphology, induced aggregation, and altered the ratio of nuclei to kinetoplasts in the parasite. The HPLC chromatograms and ATR-IR spectra of the active fractions suggested the presence of aromatic hydrocarbons with hydroxyl, carbonyl, amine, or amide functional groups. The results suggest that Z. zanthoxyloides LZT have potential chemotherapeutic effects on African trypanosomes with implications for novel therapeutic interventions in African trypanosomiasis.

Oral activity of the antimalarial endoperoxide 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Leishmania donovani complex.

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL.

Establishment of a quantitative and qualitative analysis and isolation method for tetracyclic iridoids from Morinda lucida Bentham leaves.

A new high performance liquid chromatography (HPLC) method has been established for quantitative and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Separation of 1-3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their contents were determined by measuring absorbance at 254 nm. Depending on the above system, several factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several variation on plant organs, seasonality, variation between individual trees, and branch positions within the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydrolysis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development.

Molecular characterisation of the NDM-1-encoding plasmid p2189-NDM in an Escherichia coli ST410 clinical isolate from Ghana.

Global dissemination of New Delhi metallo-ß-lactamase (NDM)-producing bacteria has become a major health threat. However, there are few reports regarding the identification and characterisation of NDM-producing bacteria from West Africa, including Ghana. An Escherichia coli strain with resistance to meropenem was isolated from the Tamale Teaching Hospital in Ghana. Its identification and determination of antibiotic susceptibility profile were carried out using commercial systems. The antibiotic resistance mechanism was analysed by phenotypic detection kits, PCR, and DNA sequencing. Conjugation experiments, S1 nuclease pulsed field gel electrophoresis, and Southern blotting were performed. Finally, the NDM-1-harbouring plasmid was characterised using next-generation sequencing and phylogenetic analysis. The meropenem-resistant Escherichia coli strain EC2189 harboured blaNDM-1 and belonged to sequence type 410. blaNDM-1 was located on the IncHI type transferrable plasmid p2189-NDM (248,807 bp long), which co-carried multiple resistance genes, such as blaCTX-M-15, aadA1, aac(6')-Ib, sul3, dfrA12, and cmlA1. p2189-NDM phylogenetically differed from previously identified blaNDM-1-positive IncHI type plasmids. A truncated Tn125 containing blaNDM-1 was bracketed by an ISSm-1-like insertion sequence upstream and by a site-specific integrase downstream. To the best of our knowledge, we have, for the first time identified and molecularly characterised an NDM-1-producing Enterobacteriaceae strain in Ghana with blaNDM-1 that had a novel genetic structure. Our findings indicate a possibility of NDM-1 dissemination in Ghana and underscore the need for constant monitoring of carbapenemase-producing bacteria.

Paenidigyamycin A, Potent Antiparasitic Imidazole Alkaloid from the Ghanaian Paenibacillus sp. DE2SH.

A new alkaloid paenidigyamycin A (1) was obtained from the novel Ghanaian Paenibacillus sp. isolated from the mangrove rhizosphere soils of the Pterocarpus santalinoides tree growing in the wetlands of the Digya National Park, Ghana. Compound 1 was isolated on HPLC at tR = 37.0 min and its structure determined by MS, 1D, and 2D-NMR data. When tested against L. major, 1 (IC50 0.75 µM) was just as effective as amphotericin B (IC50 0.31 µM). Against L. donovani, 1 (IC50 7.02 µM) was twenty-two times less active than amphotericin B (IC50 0.32 µM), reinforcing the unique effectiveness of 1 against L. major. For T. brucei brucei, 1 (IC50 0.78 µM) was ten times more active than the laboratory standard Coptis japonica (IC50 8.20 µM). The IC50 of 9.08 µM for 1 against P. falciparum 3d7 compared to artesunate (IC50 36 nM) was not strong, but this result suggests the possibility of using the paenidigyamycin scaffold for the development of potent antimalarial drugs. Against cercariae, 1 showed high anticercaricidal activity compared to artesunate. The minimal lethal concentration (MLC) and minimal effective concentration (MEC) of the compound were 25 and 6.25 µM, respectively, while artesunate was needed in higher quantities to produce such results. However, 1 (IC50 > 100 µM) was not active against T. mobilensis.

In vitro antiprotozoan activity and mechanisms of action of selected Ghanaian medicinal plants against Trypanosoma, Leishmania, and Plasmodium parasites.

Trypanosomiasis, leishmaniasis, and malaria are protozoan infections of public health importance with thousands of new cases recorded annually. Control of these infection(s) with existing chemotherapy is limited by drug toxicity, lengthy parenteral treatment, affordability, and/or the emergence of resistant strains. Medicinal plants on the other hand are used in the treatment of various infectious diseases although their chemical properties are not fully evaluated. In this study, we screened 112 crude extracts from 72 selected Ghanaian medicinal plants for anti-Trypanosoma, anti-Leishmania, and anti-Plasmodium activities in vitro and investigated their mechanisms of action. Twenty-three extracts from 20 plants showed significant antiprotozoan activity against at least 1 of 3 protozoan parasites screened with IC50 values less than 20 µg/ml. Eleven extracts showed high anti-Trypanosoma activity with Bidens pilosa whole plant and Morinda lucida leaf extracts recording the highest activities. Their IC50 (selectivity index [SI]) values were 5.51 µg/ml (35.00) and 5.96 µg/ml (13.09), respectively. Nine extracts had high anti-Leishmania activity with Annona senegalensis and Cassia alata leaf extracts as the most active. Their IC50 (SI) values were 10.8 µg/ml (1.50) and 10.1 µg/ml (0.37), respectively. Six extracts had high anti-Plasmodium activity with the leaf and stem-bark extracts of Terminalia ivorensis recording the highest activity. Their IC50 (SI) values were 7.26 µg/ml (129.36) and 17.45 µg/ml (17.17), respectively. Only M. lucida at 25 µg/ml induced significant apoptosis-like cell death in Trypanosoma parasites. Anti-Leishmania active extracts induced varying morphological changes in Leishmania parasites such as multiple nuclei and/or kinetoplast, incomplete flagella division, or nuclear fragmentation. Active extracts may be potential sources for developing new chemotherapy against these infections.

Detection of a novel putative phlebovirus and first isolation of Dugbe virus from ticks in Accra, Ghana.

Ticks are ectoparasites that transmit various types of human and animal pathogens. In particular, emerging and re-emerging diseases caused by tick-borne viruses are public health concerns around the world. However, in many countries of the sub-Saharan African region, epidemiological information on tick-borne viral infections is limited, and their prevalence and distribution remain largely unknown. In this study, we conducted surveillance on ticks to detect medically important tick-borne bunyaviruses in three study sites in and near to Accra, the capital city of Ghana, in 2015. Domestic dogs and cattle were surveyed and were found to be infested with various tick species belonging to the genera Rhipicephalus, Amblyomma and Haemaphysalis. Importantly, we detected a novel putative phlebovirus in Rhipicephalus ticks, and successfully isolated a new strain of Dugbe virus from Am. variegatum ticks. To our knowledge, this is the first report of tick-associated viruses in Ghana other than Crimean-Congo hemorrhagic fever virus.

In vitro anti-Leishmania activity of tetracyclic iridoids from Morinda lucida, benth.

Leishmaniasis is an infectious disease transmitted by the sand fly. It is caused by over 20 different species of Leishmania and has affected over 14 million people worldwide. One of the main forms of control of leishmaniasis is chemotherapy, but this is limited by the high cost and/or toxicity of available drugs. We previously found three novel compounds with an iridoid tetracyclic skeleton to have activity against trypanosome parasites. In this study, we determined the activity of the three anti-trypanosome compounds against Leishmania using field strain, 010, and the lab strain Leishmania hertigi. The minimum inhibitory concentration (MIC) of the compounds against 010 was determined by microscopy while the IC50 of compounds against L. hertigi was determined by fluorescence-activated cell sorting with Guava viacount analysis. We found two of the three compounds, molucidin and ML-F52, to have anti-Leishmania activity against both strains. The fluor-microscope observation with DAPI stain revealed that both Molucidin and ML-F52 induced abnormal parasites with two sets of nucleus and kinetoplast in a cell, suggesting that compounds might inhibit cytokinesis in Leishmania parasites. Molucidin and ML-F52 might be good lead compounds for the development of new anti-Leishmania chemotherapy.

Antitrypanosomal Activities and Mechanisms of Action of Novel Tetracyclic Iridoids from Morinda lucida Benth.

Trypanosoma brucei parasites are kinetoplastid protozoa that devastate the health and economic well-being of millions of people in Africa through the disease human African trypanosomiasis (HAT). New chemotherapy has been eagerly awaited due to severe side effects and the drug resistance issues plaguing current drugs. Recently, there has been an emphasis on the use of medicinal plants worldwide. Morinda lucida Benth. is a popular medicinal plant widely distributed in Africa, and several research groups have reported on the antiprotozoal activities of this plant. In this study, we identified three novel tetracyclic iridoids, molucidin, ML-2-3, and ML-F52, from the CHCl3 fraction of M. lucida leaves, which possess activity against the GUTat 3.1 strain of T. brucei brucei The 50% inhibitory concentrations (IC50) of molucidin, ML-2-3, and ML-F52 were 1.27 µM, 3.75 µM, and 0.43 µM, respectively. ML-2-3 and ML-F52 suppressed the expression of paraflagellum rod protein subunit 2, PFR-2, and caused cell cycle alteration, which preceded apoptosis induction in the bloodstream form of Trypanosoma parasites. Novel tetracyclic iridoids may be promising lead compounds for the development of new chemotherapies for African trypanosomal infections in humans and animals.

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene.

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.