BackgroundAntivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa.MethodsWe collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a ‘gold standard’ comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom.FindingsNone of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of ‘test’ antivenom IgGs to bind venom proteins were not substantially different from that of the ‘gold standard’ antivenoms. The least effective antivenoms had the lowest IgG content/vial.ConclusionsManufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-effica...

Transfection technology for malaria parasites provides a valuable tool for analyzing gene function and correlating genotype with phenotype. Transfection models are even more valuable when appropriate animal models are available in addition to complete in vitro systems to be able to fully analyze parasite-host interactions. Here we describe the development of such a model by using the nonhuman primate malaria Plasmodium knowlesi. Blood-stage parasites were adapted to long-term in vitro culture. In vitro-adapted parasites could readapt to in vivo growth and regain wild-type characteristics after a single passage through an intact rhesus monkey. P. knowlesi parasites, either in vitro adapted or in vivo derived, were successfully transfected to generate circumsporozoite protein (CSP) knockout parasites by double-crossover mechanisms. In vitro-transfected and cloned CSP knockout parasites were derived in a time span of only 18 days. Microscopic evaluation of developing oocysts from mosquitoes that had fed on CSP knockout parasites confirmed the impairment of sporozoite formation observed in P. berghei CSP knockout parasites. The P. knowlesi model currently is the only malaria system that combines rapid and precise double-crossover genetic manipulation procedures with complete in vitro as well as in vivo possibilities. This allows for full analysis of P. knowlesi genotype-phenotype relationships and host-parasite interactions in a system closely related to humans.The development of transfection technology for blood-stage malaria parasites (16, 23-25, 28, 29) is of great importance in the postgenomic era. It provides a direct way in which to correlate genotype with phenotype, and this enhances the further understanding of parasite biology. This will facilitate rational design of new vaccines and drugs, which are urgently needed to fight the malaria epidemic that kills annually between 1.5 and 2.7 million people, mainly young children, in Sub-Saharan Africa alone (7).Four species of Plasmodium are natural to humans (9), and two of them, Plasmodium falciparum and Plasmodium vivax, are the most prevalent and important in terms of disease. Phylogenetically, P. falciparum, the more deadly form of the two, forms a separate clade with Plasmodium reichenowi, which causes chimpanzee malaria, and P. vivax clusters with simian malarias (11). The nonhuman primate malaria, caused by Plasmodium knowlesi, a natural parasite of Macaca fascicularis, has a relatively broad host range extending to humans, where it causes a mild disease (8). The parasite is closely related to P. vivax (11), and many genes identified in P. vivax have homologues in P. knowlesi. To date, transfection techniques developed for malaria parasite blood stages (26) include episomal transfection and targeted integration with linear constructs for the rodent parasite Plasmodium berghei (24, 25), episomal transfection and targeted integration with circular DNA for the human parasite P. falciparum (28,29), and episomal transfection for the nonhuman primate malaria pa...

SummaryRed cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi . Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites.

Introduction: Infections from extended spectrum beta lactamases (ESBLs) producing enterobacteriaceae are increasingly being reported in the community setting. These infections are often multidrug resistant, with clinical and epidemiological implications, and necessitate surveillance measures based on local data. In the present study ESBLs genotypes were correlated with susceptibility to cephalosporins among ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates acquired in the community. Methodology: We investigated 28 E. coli and 24 K. pneumoniae isolates by PCR for the presence of bla SHV , bla CTX-M , and bla TEM . Minimum inhibitory concentration (MIC) for cephalosporins was determined by use of E-tests. Results: bla CTX-M was detected in 46 (88.5%), bla SHV in 13 (25%) and bla TEM in18 (34.6%) of the isolates. Nineteen (36.5%) isolates had more than one genotype detected. Urine specimens provided most of the ESBL-producing isolates (71%) followed by respiratory specimens (11%). MIC 50 for cefotaxime, ceftazidime, and ceftriaxone were at 60μg/ml, 13μg/ml, and 139μg/ml, respectively. There was a statistically significant association (p-value = 0.017) between bla SHV and resistance to ceftazidime. Though other associations could be seen among the genotypes and susceptibility profiles of the three drugs, they were not statistically significant. Twenty-four (52.2%) of the bla CTX-M isolates were sensitive and nine (19.6%) resistant to ceftazidime. For cefotaxime, 29 (63%) of bla CTX-M isolates were resistant and two (4.3%) were sensitive. Conclusion: The predominant ESBL genotype in the local community-acquired infections is bla CTX-M , most of which involved the urinary tract. ESBL genes elevated MICs for the cephalosporins, but only bla SHV could predict resistance to ceftazidime.

Experimental systems that model some of the complex interactions between parasite and host can be extremely valuable in identifying and developing new prophylactics and therapeutics against human diseases. Because primates have similar immune systems to humans, we have characterized a baboon model for understanding host response to Plasmodium knowlesi. Ten intact olive baboons (Papio anubis) of either sex were experimentally infected with P. knowlesi H strain erythrocytic parasites. The infection in these baboons was either acute or chronic. Animals with acute infection developed multiple system organ dysfunction and cerebral involvement. In chronically infected animals, only the spleen was moderately enlarged. The P. knowlesi parasitemia profile in baboons and rhesus monkeys was comparable. However, some clinical symptoms of the baboons and P. falciparum-infected humans were similar. These studies demonstrate for the first time that P. anubis is a suitable host for P. knowlesi for studying clinical symptoms and pathology.

Babesia microti-like parasites have been reported to infect captive non-human primates (NHPs). However, studies on the prevalence of Babesia spp. in free-ranging NHPs are lacking. This investigation aimed at determining the prevalence of B. microti in wild-caught Kenyan NHPs. In total, 125 animals were studied, including 65 olive baboons (Papio cynocephalus anubis) and 60 African green monkeys ([AGMs] Chlorocebus aethiops). Nested polymerase chain reaction targeting Babesia β-tubulin genes was used to diagnose infection prevalence. Results indicated a prevalence of 22% (27/125) B. microti infection in free-ranging NHPs in Kenya. There was no statistically significant difference in B. microti infection prevalence between baboons and AGMs or male and female animals. This is the first report of the presence and prevalence of B. microti in free-ranging Kenyan NHPs.

Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (Olive baboons, Papio anubis). The results showed that at a dose of 2.5 mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5 mg and 1 mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers.

This work reports the anti-plasmodial activities of Warburgia ugandensis and Zanthoxylum usambarense commonly used as phytomedicines against malaria by some Kenyan communities. AIM OF STUDY:To determine the anti-plasmodial activities of extracts from Warburgia ugandensis and Zanthoxylum usambarense against Plasmodium knowlesi and Plasmodium berghei. MATERIALS AND METHODS:Eight plant extracts were screened for in vitro anti-plasmodial activity against Plasmodium knowlesi, in a 96-well plate incubated at 37 degrees C on a RPMI culture medium supplemented with baboon serum. Of the eight, three were investigated for prophylactic and curative activities in BALB/c mice against drug-sensitive Plasmodium berghei in a 4-day test at a dose rate of 200mg/kg/day. RESULTS:Inhibitory concentrations (IC(50)) values of between 3.14 and 75 microg/ml, up to 69% chemosuppression of parasites growth and over 80% survivorship of treated mice were observed. CONCLUSION:The two medicinal plants, Warburgia ugandensis and Zanthoxylum usambarense possess bioactive compounds against malaria parasites and could be exploited for further development into malaria therapy.