Charles D. Mackenzie

Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria

The pathogenesis of fatal cerebral malaria (CM) is not well understood, in part because data from patients in whom a clinical diagnosis was established prior to death are rare. In a murine CM model, platelets accumulate in brain microvasculature, and antiplatelet therapy can improve outcome. We determined whether platelets are also found in cerebral vessels in human CM, and we performed immunohistopathology for platelet-specific glycoprotein, GPIIb-IIIa, on tissue from multiple brain sites in Malawian children whose fatal illness was severe malarial anemia, CM, or nonmalarial encephalopathy. Platelets were observed in 3 locations within microvessels: between malaria pigment and leukocytes, associated with malaria pigment, or alone. The mean surface area of platelet staining and the proportion of vessels showing platelet accumulation were significantly higher in patients with CM than in those without it. Platelet accumulation occurs in the microvasculature of patients with CM and may play a role in the pathogenesis of the disease.

Unresolved issues in anthelmintic pharmacology for helminthiases of humans.

Helminth infections are an important constraint on the health and development of poor children and adults. Anthelmintic treatment programmes provide a safe and effective response, and increasing numbers of people are benefitting from these public health initiatives. Despite decades of clinical experience with anthelmintics for the treatment of human infections, relatively little is known about their clinical pharmacology. All of the drugs were developed initially in response to the considerable market for veterinary anthelmintics in high- and middle-income countries. In contrast, the greatest burden caused by these infections in humans is in resource-poor settings and as a result there has been insufficient commercial incentive to support studies on how these drugs work in humans, and how they should best be used in control programmes. The advent of mass drug administration programmes for the control of schistosomiasis, lymphatic filariasis, onchocerciasis and soil-transmitted helminthiases in humans increases the urgency to better understand and better monitor drug resistance, and to broaden the currently very narrow range of available anthelmintics. This provides fresh impetus for developing a comprehensive research platform designed to improve our understanding of these important drugs, in order to bring the scientific knowledge base supporting their use to a standard equivalent to that of drugs commonly used in developed countries. Furthermore, a better understanding of their clinical pharmacology will enable improved therapy and could contribute to the discovery of new products.

Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.

Immunocompetence may be important in the effectiveness of Mectizan® (ivermectin) in the treatment of human onchocerciasis

Mectizan (Ivermectin) has been proved to be central to the control of onchoceriasis through self-sustainable community-based treatment. The possibility of parasitological unresponsiveness to this treatment or selection for drug resistance has emerged recently in many occasions. The reason for the reduced ability of Mectizan to maintain low levels of dermal microfilariae and early recurrent pruritus can only be speculated upon. Here, we report our own findings to address this particular issue.

Elimination of onchocerciasis from Africa: possible?

Human onchocerciasis, a parasitic disease found in 28 African countries, six Latin American countries and Yemen, causes blindness and severe dermatological problems. In 1987, efforts to control this infection shifted from vector approaches to include the mass distribution of ivermectin - a drug donated by Merck & Co. for disease control in Africa and for disease elimination in the Americas. Currently, almost 25 years later, with the Americas being highly successful and now approaching elimination, new evidence points towards the possibility of successful elimination in Africa. We suggest several major changes in the programmatic approach that through focused goal-directed effort could achieve global elimination of onchocerciasis by 2025.

Flubendazole: A candidate macrofilaricide for lymphatic filariasis and onchocerciasis field programs

A safe, f ield-usable chemotherapeutic agent that will rapidly kill adult filarial worms is urgently needed in tropical medicine. Ivermectin, distributed as Mectizan by Merck & Co. Inc., has had an enormous impact on two major human filarial infections of developing countries, onchocerciasis and lymphatic filariasis [1]. However, this agent works primarily against the microfilarial stage and lacks the ability to rapidly kill the adult parasites. Since the adult worms can survive for many years producing offspring, it has been necessary for control programs to continue drug distribution for more than a decade, for instance, until the adult worms eventually die; a labor-intensive and expensive proposition. Other agents used in filarial control programs, such as diethylcarbamazine and albendazole, may be more effective macrofilaricides than ivermectin, but for various reasons are not suitable, or are unable, to fill the role of a being rapidly acting macrofilaricide. Thus, a drug administered once, or at least in multiple doses over a very short period, that safely kills adult filarial worms would be a major contributor to the current efforts to rid the world of filarial infections and the diseases they cause. A useful field agent has typically been required to be administered in an oral dosage form, but a truly safe agent administered by another route, including parenteral approaches, could be acceptable and may even be advantageous. Given the challenges of discovery and development of agents for human use, a drug as described previously is arguably most likely, at least at present, to come from the benzimidazole group of anthelmintics. Although several benzimidazoles are currently employed in human chemotherapy, there are other potential candidate macrofilaricides in other drug classes. However, time is of the essence in finding a new drug for use in ongoing filarial control programs, and the first priority is to consider the benzimidazoles as the most likely source of a macrofilaricide. This group has provided many important effective agents for both veterinary and human medicine over the past 50 years, beginning with thiabendazole and now most prominently including albendazole and mebendazole for human parasites and a whole range of agents in veterinary medicine [2]. Benzimidazoles work by interfering with the equilibrium among tubulin subunits, tubulin and microtubules. Not surprisingly, benzimidazoles can affect host tubulin as well as that of the parasites, are typically positive in mammalian cell cytotoxicity assays and cause chromosomal nondisjunction during mitosis [3]. However, the benzimidazole anthelmintics show a differential preference for binding to nematode tubulin compared with mammalian tubulin [4], an important factor for the development of a drug against nematodes in mammals. Benzimidazoles

Detection of Circulating Parasite-Derived MicroRNAs in Filarial Infections

Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.

Interruption of Onchocerca volvulus Transmission in the Abu Hamed Focus, Sudan

Abu Hamed, Sudan, the northernmost location of onchocerciasis in the world, began community-directed treatment with ivermectin (CDTI) in 1998, with annual treatments enhanced to semiannual in 2007. We assessed the status of the parasite transmission in 2011 entomologically, parasitologically, and serologically. O-150 pool screening showed no parasite DNA in 17,537 black flies collected in 2011 (95% confidence interval upper limit [95% CI UL] = 0.023). Skin microfilariae, nodules, and signs of skin disease were absent in 536 individuals in seven local communities. Similarly, no evidence of Onchocerca volvulus Ov16 antibodies was found in 6,756 school children ≤ 10 years (95% CI UL = 0.03%). Because this assessment of the focus meets the 2001 World Health Organization (WHO) criteria for interrupted transmission, treatment was halted in 2012, and a post-treatment surveillance period was initiated in anticipation of declaration of disease elimination in this area. We provide the first evidence in East Africa that long-term CDTI alone can interrupt transmission of onchocerciasis.

Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution.

Introduction: A clinically significant endemic focus of onchocerciasis existing in Esmeraldas Province, coastal Ecuador has been under an ivermectin mass drug administration program since 1991. The main transmitting vector in this area is the voracious blackfly, Simulium exiguum. This paper describes the assessments made that support the decision to cease mass treatment. Methodology and Principle Findings: Thirty-five rounds of ivermectin treatment occurred between 1991–2009 with 29 of these carrying >85% coverage. Following the guidelines set by WHO for ceasing ivermectin distribution the impact on parasite transmission was measured in the two vector species by an O-150 PCR technique standard for assessing for the presence of Onchocerca volvulus. Up to seven collection sites in three major river systems were tested on four occasions between 1995 and 2008. The infectivity rates of 65.0 (CI 39–101) and 72.7 (CI 42–116) in 1995 dropped to zero at all seven collection sites by 2008. Assessment for the presence of antibodies against O. volvulus was made in 2001, 2006, 2007 and 2008 using standard ELISA assays for detecting anti-Ov16 antibodies. None of total of 1810 children aged 1–15 years (between 82 and 98% of children present in the surveyed villages) tested in the above years were found to be carrying antibodies to this antigen. These findings were the basis for the cessation of mass drug treatment with ivermectin in 2009. Significance: This fulfillment of the criteria for cessation of mass distribution of ivermectin in the only known endemic zone of onchocerciasis in Ecuador moves the country into the surveillance phase of official verification for national elimination of transmission of infection. These findings indicate that ivermectin given twice a year with greater than 85% of the community can move a program to the final stages of verification of transmission interruption.

Progress and challenges in the discovery of macrofilaricidal drugs

Control of human filarial infections currently depends on chemotherapeutic strategies predominantly directed at microfilariae. Doxycycline therapy in an extended daily dose regimen sterilizes and kills adult stages, but the utility of this drug for routine field use remains an issue of concern. No macrofilaricidal drugs with efficacy after one or two doses are available for use, delaying the achievement of the elimination or eradication of onchocerciasis and lymphatic filariasis. Moxidectin, a macrocyclic lactone, is currently in clinical trials for onchocerciasis. A few other drugs that have already been approved for use in veterinary practice or in human medicine for other indications are available for investigation. Early drug discovery pipelines are poorly populated and the process of macrofilaricide discovery and development remains highly challenging. In particular, the lack of convenient, validated animal models in an antifilarial drug discovery pathway is an unresolved issue.