John Kagira

Efficacy of the diamidine DB75 and its prodrug DB289, against murine models of human African trypanosomiasis.

The choice of drugs for the treatment of sleeping sickness is extremely limited. To redress this situation, the recently synthesised diamidine, 2,5-bis(4-amidinophenyl)-furan (DB75, furamidine) and its methamidoxime prodrug, 2,5-bis(4-amidinophenyl)-furan-bis-O-methylamidoxime (DB289, pafuramidine) were, together with pentamidine, evaluated for efficacy in acute rodent models. The activity was compared in three common mouse models that mimic the first stage of human African trypanosomiasis. The mice were infected with the pleomorphic T .b. rhodesiense strains KETRI2537 and STIB900 or with the monomorphic T. b. brucei strain STIB795. Importantly, DB75 showed activity superior to that of pentamidine at comparable doses in all three mouse models. Complete cures were achieved with oral dosing of the prodrug DB289 in all three models without any overt toxicity. This shows that the prodrug strategy was successful in terms of reducing toxicity and increasing efficacy and oral bioavailability.

Questionnaire Survey on the Occurrence of Risk factors for Toxoplasma gondii infection amongst Farmers in Thika District, Kenya

A survey was conducted to determine the occurrence of risk factors for Toxoplasma gondii infection amongst farmers in Thika District, Kenya. Interviews were conducted in a total of 385 households using a structured questionnaire. The water consumed at household level originated from taps (74.3%), rivers or streams (15.1%), wells (5.4%) and boreholes (5.2%). A number of households (46.8%) consumed water without boiling or applying any form of treatment. All respondents washed vegetables before cooking, whilst 99.0% washed fruits before eating. Boiled milk was preferred by 99.5% of the farmers. The majority (85.2%) consumed beef more often, whilst 1.6% consumed pork. The majority (98.7%) consumed thoroughly cooked meat. Meat was preserved by 17% of farmers. Only four farmers (1.2%) who practised mixed farming used gloves when handling livestock manure. Five farmers (1.6%) reported the occurrence of abortion in ruminants and pigs on their farms within the last two years before the study. Almost half (44.9%) of the households owned cats, which were kept mainly as pets (79.8%) and for deterring rodents (20.2%). The majority of households (91.3%) fed the cats on leftovers, whilst 8.1% fed cats with raw offal. Sixteen households (9.2%) provided housing for cats. Only five households (2.8%) had litter boxes, but none of the households with litter boxes used gloves when cleaning them out. Disposal of cat faeces was done mainly by women (55.5%). Only one farmer (0.3%) had some knowledge about toxoplasmosis, but was not aware of the transmission mechanism. The study highlights the need for public health education to raise awareness of risk factors for toxoplasmosis.

Lipid metabolism and other metabolic changes in vervet monkeys experimentally infected with Trypanosoma brucei rhodesiense

Backgroundu2002 Human African trypanosomiasis is associated with metabolic changes which have not been well characterized.

IgM, lgG and IL-6 profiles in the Trypanosoma brucei brucei monkey model of human African trypanosomiasis

Human African trypanosomiasis (HAT) patients manifest immunological profiles, whose variations over time can be used to indicate disease progression. However, monitoring of these biomarkers in human patients is beset by several limitations which can be offset by using chronic animal models. A recent improved monkey model of HAT using a Trypanosoma brucei brucei isolate has been developed but the immunological profile has not been elucidated. The objectives of the current study was to determine the IgM, IgG and IL-6 profiles in blood and cerebrospinal fluid (CSF) in vervet monkeys infected with T. b. brucei. Three vervet monkeys were infected intravenously with 105T. b. brucei, monitored for disease development and subsequently treated 28days post infection (dpi) sub-curatively using diminazene aceturate (DA) to induce late stage disease and curatively treated with melarsoprol (Mel B) at 119 dpi, respectively. Matched serum and cerebrospinal fluid (CSF) samples were obtained at regular intervals and immunospecific IgM, immunoglobulin G (IgG) were quantified by ELISA while IL-6 was assayed using a cytometric bead array (CBA) kit. Results showed that following infection, CSF IgM, IgG, IL-6 and serum IL-6 were significantly (p<0.05) elevated with peak levels coinciding with relapse parasitaemia. The IgG levels increased to reach OD peak levels of 0.442±0.5 at 126 dpi. After curative treatment with MelB, the serum IgM and Ig G levels fell rapidly to attain pre-infection levels within 35 and 49days, respectively. This shows that the profile of these immunoglobulins can be used as an indicator of curative treatment. CSF IL-6 concentrations of infected vervet monkeys showed no significant change (P>0.05) between infection and 35 dpi but levels increased significantly (P<0.05) with the highest level of 55.53pg/ml recorded at112 dpi. IL-6 elevation from 35 dpi may be indicative of parasite neuroinvasion hence can be used as possible candidate marker for late stage disease in the monkey model. Further, the marker can also be used in conjunction with IgG and IgM as markers for development of test of cure for HAT.

Use of the nested polymerase chain reaction for detection of Toxoplasma gondii in slaughterhouse workers in Thika District, Kenya

BACKGROUNDnThe widely used methods of diagnosis of Toxoplasma gondii are serological. Current reports indicate a high seroprevalence of T. gondii in humans in Kenya. There is a need for more sensitive diagnostic tests, especially when the specific antibody titres are below detectable threshold levels. Use of the polymerase chain reaction (PCR) targeting the repetitive 529 base pair loci has been reported to be sensitive and specific.nnnOBJECTIVEnTo detect T. gondii in a high-risk group of public health workers in Thika District, Kenya.nnnMETHODSnIn total, 87 human blood samples were collected from male slaughterhouse workers between xa01 March 2013 and xa025 June 2013. The DNA extracted was amplified by the nested PCR.nnnRESULTSnT. gondii was detected in 39.1% (34/87) of the workers. In the cow-sheep-goat slaughterhouses the prevalence ranged between 20% and 60%, while all the chicken slaughterhouse workers (6/6, 100%) tested positive. The difference in T. gondii positivity between the workers in the chicken slaughterhouse and those in the cattle-sheep-goat slaughterhouses was statistically significant (p=0.003).nnnCONCLUSIONnThis study shows the presence of T. gondiiin an asymptomatic high-risk group in Thika District, indicating the need for enhancement of public health awareness.

Comparative pathogenicity of Trypanosoma brucei rhodesiense strains in Swiss white mice and Mastomys natalensis rats.

We evaluated Mastomys natelensis rat as an animal model for Rhodesian sleeping sickness. Parasitaemia, clinical and pathological characteristics induced by T. b. rhodesiense isolates, KETRI 3439, 3622 and 3637 were compared in Mastomys rats and Swiss white mice. Each isolate was intra-peritonially injected in mice and rat groups (n=12) at 1×10(4) trypanosomes/0.2mL. Pre-patent period (PP) range for KETRI 3439 and KETRI 3622-groups was 3-6 days for mice and 4-5 days for rats while for KETRI 3637-infected mice and rats was 5-9 and 4-12 days, respectively. Pairwise comparison between PP of mice and rats separately infected with either isolate showed no significant difference (p>0.05). The PPs of KETRI 3637-infected mice were significantly (p>0.01) longer than those infected with KETRI 3439 or KETRI 3622, a trend also observed in rats. The second parasitaemic wave was more prominent in mice. Clinical signs included body weakness, dyspnoea, peri-orbital oedema and extreme emaciation which were more common in rats. Survival time for KETRI 3439 and 3622-infected groups was significantly (p<0.05) longer in mice than rats but similar in KETRI 3637-infected groups. Inflammatory lesions were more severe in rats than mice. All mice and KETRI 3622-infected rats had splenomegaly, organ congestion with rats additionally showing prominent lymphadenopathy. KETRI 3439-infected rats showed hemorrhagic pneumonia, enteritis with moderate splenomegaly and lymphadenopathy. KETRI 3637-infected rats had the most severe lesions characterized by prominent splenomegaly, lymphadenopathy, hepatomegaly, enlarged adrenal glands, organ congestion, generalized oedemas, gastroenteritis, pneumonia and brain congestion. KETRI 3637-infected Mastomys is a suitable model for studying pathophysiology of HAT.

Erythrina abyssinica prevents meningoencephalitis in chronic Trypanosoma brucei brucei mouse model

Human African trypanosomiasis is prevalent in Sub-sahara African countries that lie between 14° North and 29° south of the equator. Sixty million people are at risk of infection. Trypanosoma brucei gambesience occurs in West and Central Africa while Trypanosoma brucei rhodesience occurs in East and Southern Africa. The neurological stage of the disease is characterized by neuroinflammation. About 10xa0% of patients treated with the recommended drug, melarsoprol develop post treatment reactive encephalopathy, which is fatal in 50xa0% of these patients, thus melarsoprol is fatal in 5xa0% of all treated patients. This study was aimed at establishing the potential activity of Erythrina abyssinica in reducing neuroinflammation following infection with Trypanosoma brucei brucei. Swiss white mice were divided into ten groups, two control groups and eight infected groups. Infected mice received either methanol or water extract of Erythrina abyssinica at 12.5, 25, 50 or 100xa0mg/kg body weight. Parasite counts were monitored in peripheral circulation from the third day post infection up to the end of the study. Brains were processed for histology, immunohistochemistry scanning and transmission electron microscopy. Following infection, trypanosomes were observed in circulation 3xa0days post-infection, with the parasitaemia occurring in waves. In the cerebrum, typical brain pathology of chronic trypanosomiasis was reproduced. This was exhibited as astrocytosis, perivascular cuffing and infiltration of inflammatory cells into the neuropil. However, mice treated with Erythrina abyssinica water extract exhibited significant reduction in perivascular cuffing, lymphocytic infiltration and astrocytosis in the cerebrum. The methanol extract did not have a significant difference compared to the non-treated group. This study provides evidence of anti-inflammatory properties of Erythrina abyssinica and may support its wide use as a medicinal plant by various communities in Kenya.

Trypanosoma brucei gambiense infection in vervet monkeys: a potential model for early‐stage disease

Non‐human primates are important experimental models for human African trypanosomiasis.

Development of a safer laboratory vervet monkey model for the study of human African trypanosomiasis

Background There are three subspecies of Trypanosoma brucei: T. b. gambiense, T. b. rhodesiense and T. b. brucei. The first two are infectious to humans, whilst T. b. brucei is not. Identifying an animal model of T. b. brucei that mimics human African trypanosomiasis (HAT) would enable researchers to study HAT without subjecting themselves to undue risks such as accidental infection. Objectives This study assessed the sequential clinical, parasitological and haematological changes in vervet monkeys infected with T. b. brucei. Methods Three vervet monkeys were infected with a 104 inoculum of T. b. brucei (isolate GUTat 1). Late-stage disease was induced by subcurative treatment with diminazene aceturate 28 days post-infection. The animals were treated curatively with melarsoprol upon relapse. Parasitaemia and clinical signs were monitored daily and, at weekly intervals, the monkeys’ blood and cerebrospinal fluid (CSF) were sampled for haematology and parasitosis assessments, respectively. Results The first-peak parasitaemia was observed between seven and nine days post-infection. Clinical signs associated with the disease included fever, dullness, pallor of mucous membranes, lymphadenopathy, splenomegaly and oedema. Late-stage signs included stiffness of joints and lethargy. The monkeys developed a disease associated with microcytic hypochromic anaemia. There was an initial decline, followed by an increase, in total white blood cell counts from early- to late-stage disease. Trypanosomes were detected in the CSF and there was a significant increase in white cell counts in the CSF during late-stage disease. Infected vervet monkeys displayed classical clinical symptoms, parasitological and haematological trends that were similar to monkeys infected with T.b. rhodesiense. Conclusion The T. b. brucei vervet monkey model can be used for studying HAT without putting laboratory technicians and researchers at high risk of accidental infection.

Socioeconomic and health implications of human–wildlife interactions in Nthongoni, Eastern Kenya

The human population in Kenya has doubled over the last 25 years and is expected to rise two fold by 2050. Thus, pressure for human space has led to encroachment into wildlife habitats, increasing human—wildlife interactions. Such interactions pose serious health risks to both humans and wildlife, yet studies to understand these risks are limited in Kenya. To understand the possible predisposing factors for zoonoses at the human—wildlife interface, a survey was carried out in Nthongoni, an area bordering Tsavo and Chyulu Hills national parks in Kenya. Questionnaires were administered to 11 key informants and 200 residents from 35 villages. Our results indicate that the majority (75%) of the respondents suffered from crop raids and livestock depredation by wildlife. On their part, residents killed wildlife for: subsistence (41%), revenge (35%), bush-meat trade (22%), and other undisclosed reasons. Nineteen per cent of the respondents were knowledgeable about disease transmission through bush-meat. Qualitative data revealed helplessness, bitterness and revenge tendencies by farmers due to wildlife losses, which contributed to their poverty. This study enhances our understanding of human—wildlife interactions and the associated socioeconomic, health and conservation implications. It demonstrates the predicaments communities living adjacent to wildlife areas face and the need to involve them in sustainable management of the areas. We recommend identification of appropriate alternative livelihoods, to mitigate illegal bush-meat and agricultural practices that attract wildlife, leading to conflicts. In addition, responsive health and conservation education, and participatory research aimed at advising policy, are necessary to cushion the communities from wildlife damages.