The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Author Summary

The search for new drugs for human neglected diseases accelerated in the past decade, based on the recognition that addressing these infections was necessary for global poverty reduction. The expansion of discovery and development programmes was supported by donor investment, increasing participation of the industry and the creation of Product Development Partnership (PDP) enterprises. Despite these efforts, major discovery gaps remain as, apart from some repurposed drugs and a few new molecules for malaria, no new candidate has been recently transitioned from discovery into development for the major Neglected Tropical Diseases (NTDs). In this publication, we present a collaborative network model for drug discovery based on coordinated North-South partnerships. This network carried out low-to-medium throughput whole-organism screening assays against seven NTDs (malaria, leishmaniasis, human African trypanosomiasis [HAT], Chagas' disease, schistosomiasis, onchocerciasis and lymphatic filariasis) together with an early assessment of compound toxicity in mammalian cells. We describe a screening campaign of 10,000 molecules, its outcome and the implications of this strategy for enhancing the efficiency and productivity of drug discovery for NTDs.

Background

Data on the levels of resistance of Mycobacterium tuberculosis complex (MTBC) strains to first line anti-tuberculosis drugs in Cameroon, and on the species of MTBC circulating in the country are obsolete. The picture about 10 years after the last studies, and 6 years after the re-organisation of the National Tuberculosis (TB) Control Programme (NTBCP) is not known.

Methods

The study was conducted from February to July 2009 in the West and Centre regions of Cameroon. A total of 756 suspected patients were studied. MTBC species were detected by the standard Ziehl-Neelsen staining method. Bacterial susceptibility to the first line drugs [isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (SM)] were performed on cultures using the indirect proportion method. MTBC species were identified by standard biochemical and culture methods.

Results

Of the 756 suspected patients, 154 (20.37%) were positive by smear microscopy. Of these, 20.77% were HIV patients. The growth of Mycobacterium was observed with the sputa from 149 (96.75%) subjects. All the isolates were identified as either M. tuberculosis or M. africanum. Among these, 16 (10.73%) were resistant to at least one drug (13.3% for the West region and 8.1% for the Centre). The initial resistance rates were 7.35% for the Centre region and 11.29% for the West region, while the acquired resistance rates were 16.66% (1/6) for the Centre region and 23.07% (3/13) for the West. Within the two regions, the highest total resistance to one drug was obtained with INH and SM (2.68% each). Multidrug-resistance (MDR) was observed only in the West region at a rate of 6.67%. No resistance was recorded for EMB.

Conclusions

M. tuberculosis and M. africanum remain the MTBC species causing pulmonary TB in the West and Centre regions of Cameroon. Following the re-organisation of the NTBCP, resistance to all first line anti-TB drugs has declined significantly (p < 0.05 for West; and p < 0.01 for Centre) in comparison to previous studies. However, the general rates of anti-TB drug resistance remain high in the country, underscoring the need for greater enforcement of control strategies.

Background

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.

Methods

Sixteen crude extracts were prepared from various parts of M. discoidea and H. africanum using different organic solvents. The filaricidal activities were determined in vitro. Cytotoxicity of the active extracts was assessed on monkey kidney epithelial cells in vitro and the selectivity indices (SI) of the extracts determined. Acute toxicity of the promising extracts was investigated in mice.

Results

Four out of the 16 extracts showed microfilaricidal activity based on motility reduction, whereas, none showed macrofilaricidal activity based on the MTT/formazan assay. The methylene chloride extract of H. africanum leaves (HLC) recorded the lowest IC50 of 31.25 μg/mL and an IC100 of 62.5 μg/mL. The SI for the active extracts ranged from 0.5 - 2.63. No form of acute toxicity was observed in mice. Phytochemical analysis revealed the presence of anthraquinones, sterols and terpenoids in the promising extracts.

Conclusions

The non-polar extracts of M. discoidea and H. africanum are potential sources of new microfilaricidal lead compounds, and the results support their use in traditional medicine.

Background

Despite considerable efforts, a suitable vaccine against Onchocerca volvulus infection has remained elusive. Herein, we report on the use of molecular tools to identify and characterize O. volvulus antigens that are possibly associated with the development of concomitant immunity in onchocerciasis.

Methodology/Principal Findings

Third-stage larvae (L3) and molting L3 (mL3) O. volvulus stage-specific cDNA libraries were screened with a pool of sera from chronically infected patients who had likely developed such immunity. The 87 immunoreactive clones isolated were grouped into 20 distinct proteins of which 12 had already been cloned and/or characterized before and 4 had been proven to be protective in a small O. volvulus animal model. One of these, onchocystatin (Ov-CPI-2), a previously characterized O. volvulus cysteine proteinase inhibitor was, overall, the most abundant clone recognized by the immune sera in both the L3 and mL3 cDNA libraries. To further characterize its association with protective immunity, we measured the IgG subclass and IgE class specific responses to the antigen in putatively immune (PI) and infected (INF) individuals living in a hyperendemic area in Cameroon. It appeared that both groups had similar IgG3 and IgE responses to the antigen, but the INF had significantly higher IgG1 and IgG4 responses than the PI individuals (p<0.05). In the INF group, the IgG3 levels increased significantly with the age of the infected individuals (r = 0.241; p<0.01). The IgG1 responses in the INF were high regardless of age. Notably, culturing L3 in vitro in the presence of anti-Ov-CPI-2 monospecific human antibodies and naïve neutrophils resulted in almost complete inhibition of molting of L3 to L4 and to cytotoxicity to the larvae.

Conclusions/Significance

These results add to the knowledge of protective immunity in onchocerciasis and support the possible involvement of anti-Ov-CPI-2 IgG1 and/or IgG3 cytophilic antibodies in the development of protective immunity in the PI and the INF. The results further support the consideration of Ov-CPI-2 as a leading target for an anti-L3 vaccine.

Author Summary

Onchocerciasis is a chronic and highly debilitating disease of humans caused by a worm called Onchocerca volvulus. This worm can live in the human body for over 15 years. The disease affects mainly the skin and eyes and is the second leading infectious cause of blindness worldwide. There is currently no vaccine to prevent the infection. Available drugs can give short-term relief but cannot cure the infection. To prevent infection, a vaccine against the third-stage infective larva, L3, or the developing larva is required. These stages were shown to be the targets of protective immunity that develops in individuals who live in onchocerciasis endemic regions. One type of protective immunity has been shown to develop with age and is called concomitant immunity. In the present study, we have identified a number of larval antigens that may be associated with the development of such immunity. The most prominent of these antigens was Ov-CPI-2, also called onchocystatin, which had previously been shown to be a promising vaccine candidate. This antigen was further characterized and confirmed to be possibly also a target of immune protection that develops in the infected individuals with age and is referred to as concomitant immunity.