Trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human African trypanosomiasis

We have investigated the pathogenicity of tsetse (Glossina pallidipes)-transmitted cloned strains of Trypanosoma brucei rhodesiense in vervet monkeys. Tsetse flies were confirmed to have mature trypanosome infections by xenodiagnosis, after which nine monkeys were infected via the bite of a single infected fly. Chancres developed in five of the nine (55.6%) monkeys within 4 to 8 days post infection (dpi). All nine individuals were successfully infected, with a median pre-patent period of 4 (range = 4-10) days, indicating that trypanosomes migrated from the site of fly bite to the systemic circulation rapidly and independently of the development of the chancre. The time lag to detection of parasites in cerebrospinal fluid (CSF) was a median 16 (range = 8-40) days, marking the onset of central nervous system (CNS, late) stage disease. Subsequently, CSF white cell numbers increased above the pre-infection median count of 2 (range = 0-9) cells/microl, with a positive linear association between their numbers and that of CSF trypanosomes. Haematological changes showed that the monkeys experienced an early microcytic-hypochromic anaemia and severe progressive thrombocytopaenia. Despite a 3-fold increase in granulocyte numbers by 4 dpi, leucopaenia occurred early (8 dpi) in the monkey infection, determined mainly by reductions in lymphocyte numbers. Terminally, leucocytosis was observed in three of nine (33%) individuals. The duration of infection was a median of 68 (range = 22-120) days. Strain and individual differences were observed in the severity of the clinical and clinical pathology findings, with two strains (KETRI 3741 and 3801) producing a more acute disease than the other two (KETRI 3804 and 3928). The study shows that the fly-transmitted model accurately mimics the human disease and is therefore a suitable gateway to understanding human African trypanosomiasis (HAT; sleeping sickness).     

Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal

Background

The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (α-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice.

Methodology and results

Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples.

Conclusions/significance

The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.     

赞比亚的人类发展与环境挑战

由于赞比亚社会经济及人民生活水平较低,赞比亚人民为了生存,不得不依靠掠夺性地开发自然生态系统和破坏环境来获得资源。赞比亚有60%以上的人民是农村居民,直接靠自然环境资源维持生存,对当地的自然环境造成了很大的影响。这种人类对环境的压力包括森林的毁坏,农田覆土的不断流失,土壤侵蚀以及气候的多变性造成的周期性干旱等。而人口的快速增长,当地社会经济的长期疲软以及大气、水、土地资源的污染更是给原本脆弱的生态环境雪上加霜。本文受统计数据的可获得性限制,部分结论尚未定量化,但其反映的人类发展与环境保护之间的矛盾却是客观存在的。     

Analysis of oxidative signalling induced by ozone in Arabidopsis thaliana

We are using acute ozone as an elicitor of endogenous reactive oxygen species (ROS) to understand oxidative signalling in Arabidopsis. Temporal patterns of ROS following a 6 h exposure to 300 nL L(-1) of ozone in ozone-sensitive Wassilewskija (Ws-0) ecotype showed a biphasic ROS burst with a smaller peak at 4 h and a larger peak at 16 h. This was accompanied by a nitric oxide (NO) burst that peaked at 9 h. An analysis of antioxidant levels showed that both ascorbate (AsA) and glutathione (GSH) were at their lowest levels, when ROS levels were high in ozone-stressed plants. Whole genome expression profiling analysis at 1, 4, 8, 12 and 24 h after initiation of ozone treatment identified 371 differentially expressed genes. Early induction of proteolysis and hormone-responsive genes indicated that an oxidative cell death pathway was triggered rapidly. Down-regulation of genes involved in carbon utilization, energy pathways and signalling suggested an inefficient defense response. Comparisons with other large-scale expression profiling studies indicated some overlap between genes induced by ethylene and ozone, and a significant overlap between genes repressed by ozone and methyl jasmonate treatment. Further, analysis of cis elements in the promoters of ozone-responsive genes also supports the view that phytohormones play a significant role in ozone-induced cell death.     

Correlation mapping method for generating microcirculation morphology from optical coherence tomography (OCT) intensity images

Standard optical coherence tomography (OCT) in combination with software tools can be harnessed to generate vascular maps in vivo. In this study we have successfully combined a software algorithm based on correlation statistic to reveal microcirculation morphology on OCT intensity images of a mouse brain in vivo captured trans‐cranially and through a cranial window. We were able to estimate vessel geometry at bifurcation as well as along vessel segments down‐to mean diameters of about 24 μm. Our technique has potential applications in cardiovascular‐related parameter measurements such as volumetric flow as well as in assessing vascular density of normal and diseased tissue. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Colonization of woody seedlings in the understory of actively and passively restored tropical moist forests

The status of woody seedling colonization gives clues about the self‐sustainability of restored forests, a tenet of restoration success. Little is known about woody seedling colonization in restored afrotropical forests. We evaluated effects of restoration methods (active vs. passive), sampling year, restoration age, and distance from old‐growth forests on seedling colonization in restored afrotropical moist forests. Seedlings were measured in 2011 and 2014 in 71 clusters of 284 permanent sampling plots (12.6 m² each) in actively (initially 3–16 years old) and 21 clusters of 63 plots in passively restored forests (initially 16 years old) in Kibale National Park, western Uganda. Seedlings were also measured in nearby old‐growth forests in three clusters of five plots in 2014. We determined species diversity, richness, abundance per plot, and species composition as measures of seedling colonization in restored and old‐growth forests. We found that diversity, richness, and abundance of seedlings were significantly higher in passively than actively restored forests. Diversity and richness but not abundance significantly increased between sampling years and with restoration age. Distance from old‐growth forests did not significantly affect diversity, richness, and abundance. Species composition of actively and passively restored forests was different from that of old‐growth forests after 19 years since restoration started. Our results show that passive restoration should be the preferred method for recovering afrotropical forests, and highlight the effect of continued management on biodiversity of restored forests.     

The Prevalence of Tuberculosis in Zambia: Results from the First National TB Prevalence Survey, 2013–2014

Background

Tuberculosis in Zambia is a major public health problem, however the country does not have reliable baseline data on the TB prevalence for impact measurement; therefore it was among the priority countries identified by the World Health Organization to conduct a national TB prevalence survey

Objective

To estimate the prevalence of tuberculosis among the adult Zambian population aged 15 years and above, in 2013–2014.

Methods

A cross-sectional population-based survey was conducted in 66 clusters across all the 10 provinces of Zambia. Eligible participants aged 15 years and above were screened for TB symptoms, had a chest x-ray (CXR) performed and were offered an HIV test. Participants with TB symptoms and/or CXR abnormality underwent an in-depth interview and submitted one spot- and one morning sputum sample for smear microscopy and liquid culture. Digital data collection methods were used throughout the process.

Results

Of the 98,458 individuals who were enumerated, 54,830 (55.7%) were eligible to participate, and 46,099 (84.1%) participated. Of those who participated, 45,633/46,099 (99%) were screened by both symptom assessment and chest x-ray, while 466/46,099 (1.01%) were screened by interview only. 6,708 (14.6%) were eligible to submit sputum and 6,154/6,708 (91.7%) of them submitted at least one specimen for examination. MTB cases identified were 265/6,123 (4.3%). The estimated national adult prevalence of smear, culture and bacteriologically confirmed TB was 319/100,000 (232-406/100,000); 568/100,000 (440-697/100,000); and 638/100,000 (502-774/100,000) population, respectively. The risk of having TB was five times higher in the HIV positive than HIV negative individuals. The TB prevalence for all forms was estimated to be 455 /100,000 population for all age groups.

Conclusion

The prevalence of tuberculosis in Zambia was higher than previously estimated. Innovative approaches are required to accelerate the control of TB.     

Comparing seed removal rates in actively and passively restored tropical moist forests

High rates of seed removal can impede forest recovery, but tropical seed removal studies are few and mainly from the neotropics. Little is known about the comparative influences of active restoration (i.e. planting) and passive restoration (i.e. protection of natural regrowth) on seed removal. We conducted an evaluation of seed removal in grasslands, natural forests (tropical moist semideciduous forest), and actively (21‐, 17‐, 16‐, 11‐, 8‐, and 6‐year‐old) and passively (21‐year‐old) restored forests in Kibale National Park, Uganda. We wanted to compare the effect of vegetation type, time since restoration and restoration actions (i.e. active vs. passive) on removal of seeds of five animal‐dispersed tree species during wet and dry seasons. Seeds were either fully exposed or placed in closed mesh cages or under a mesh roof. We used differential removal rates between these treatments to attribute seed removal to different animal taxa. Seed removal rate (percentage of seed removed over a 4‐day period) was highest in passively restored forests, compared with actively restored forests, grasslands, and natural forests. We detected no significant relationship between time since restoration and seed removal rates within actively restored sites. Seed removal rate from roofed treatments was not significantly different from removal from open treatments but was significantly higher than removal from closed treatments, which we interpret as reflecting the greater effect of small mammals versus insects. Smaller seeds tended to be removed at a greater rate than larger seeds. We discuss the implications of these findings for forest regeneration.