Enabling the Development and Deployment of Next Generation Point-of-Care Diagnostics

A major goal of the 1st International Point-of-Care Diagnostic Workshop in Nairobi, Kenya was to provide a forum for open dialog concerning current challenges in, and potential solutions for, the development of the next generation of POC diagnostics. The focus was not solely on descriptions of new technologies in development but also included demonstrations of their use in a field setting. The resulting conversations identified a number of obstacles to the successful translation of prototypes into field-deployable tools. These obstacles superseded those typically encountered in research; changes must be implemented at the institutional and governmental level to enable equitable collaborations between Western and African partners, and proper funding mechanisms must be established to support these collaborations. Additionally, this workshop showcased emerging technologies for POC tests and fostered new partnerships between technology developers and African research laboratories. Equitable partnerships are critical for the successful implementation of new POC technology. The attendees agreed that the most effective methods to effect change require improved communication of needs, ideas and abilities, and a conduit for the sharing of experiences and information. We plan to implement many of the changes that are suggested here in our own research programs and to use future conferences and workshops to guide the development of both technologies and partnerships. Our successes and failures will serve as models for those scientists striving to develop technological and biomedical solutions to similar problems in global health.

Human African trypanosomiasis (HAT) is a disease of poor rural communities caused by extracellular protozoan parasites of the genus Trypanosoma. In early or Stage 1 infection when parasites are in blood and the lymphatic system, treatment is relatively safe and cheap. During this time however, clinical signs are not suggestive of HAT, and diagnostic tests have problems of sensitivity and specificity. Many cases therefore remain undetected and parasites invade the brain, resulting in late or Stage 2 disease, and symptoms associated with HAT are observed. Treatment of Stage 2 HAT is lengthy and expensive, and the drugs used can cause potentially fatal side effects. Unfortunately, tests to determine the stage of disease are non-specific and insensitive. Early and accurate diagnosis and staging of HAT would ensure safe treatment, reduced transmission and accelerated control of the disease. Major steps in development of novel tests for diagnosis of HAT, and for determining the stage of disease have been made recently. A simple rapid test for screening populations is undergoing evaluation. Individuals found positive with this test will be confirmed by demonstration of parasites using an LED fluorescence microscope, or by a simple molecular method called loop-mediated isothermal amplification (LAMP) of DNA. Biomarkers that accurately distinguish Stage 1 and 2 disease have also been identified in spinal fluid of HAT patients. Completion of development of these new tests and their introduction for diagnosis of HAT is likely to accelerate control and the eventual elimination of the disease.

Dr. Paul Yager University of Washington, WA, USA
Dr. Yager received his A.B. in Biochemistry from Princeton (1975), and a Ph.D. in Chemistry from the University of Oregon (1980). He was an NRC Fellow at the Naval Research Laboratory in DC from 1980 to 1982, joining the NRL staff in 1982. He joined the Department of Bioengineering at the University of Washington in 1987, and became Chair in 2008.
For the last two decades, work in the lab has focused on development of microfluidic devices for monitoring of medically-significant analytes. The primary goal of current work is increasing access to healthcare through decentralization of biomedical diagnostic testing. A grant from the Bill & Melinda Gates Foundation developed a low-cost point-of-care platform for diagnosing diseases in the developing world, now being developed by Micronics, Inc. Since 2008, the lab has focused on instrument-free diagnostics based on 2-dimensional paper networks. Specifics can be found at http://faculty.washington.edu/yagerp/.

Paul Yager, Elain Fu, Barry Lutz
Department of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195, USA Email: yagerp@uw.edu Two-dimensional paper networks (2DPNs) allow complex chemical processing in a very low-cost format. We have, for the last 4 years, been learning how to translate what we have learned about point-of-care diagnostic technologies in conventional microfluidics into the language of porous media. The wicking of fluids in porous materials (like paper, nitrocellulose membranes, etc.) allows us to discard pumps, which allows great savings in complexity, and the potential to perform complex tests without any permanent instruments. However, there are many physical and chemical differences between open ducts and porous media. We have put a good deal of effort into understanding the performance and design rules of simple paper systems. Currently, the primary applications for this technology in our lab are highly-sensitive multiplexed immunoassays and multiplexed isothermal nucleic acid amplification assays. All assays are designed with visible optical readout that can be captured and quantified using camera-equipped cellular phones.

Dr. Marya Lieberman University of Notre Dame, IN, USA
Marya Lieberman is an associate professor in the Department of Chemistry and Biochemistry at the University of Notre Dame in South Bend, Indiana. Her research is in the area of surface chemistry, selfassembly of two-and three-dimensional structures, and molecular electronics. A new project focuses on detection of counterfeit drugs with paper-based sensors and cell phone imaging; this technology is currently being field tested in Eldoret, Kenya. Abstract:

Marya Lieberman
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556,USA Email: mlieberm@nd.edu We are developing inexpensive analytical devices to detect fake pharmaceuticals. The devices are paper-based, contain all necessary reagents, do not require power, and can be read by taking a picture with a cell phone and sending it to a web site. These fast screening tests can single out suspicious drugs for confirmatory testing and greatly reduce the cost of monitoring critical pharmaceuticals. The fabrication method involves thermal wax printing and deposition of reagents using a micropipette or a device called a "frog". Image analysis methods will be discussed & shared.
Paper-based fluidic devices constitute a promising platform for point-of-care diagnostic devices. A feature common to all these devices is that the top and bottom faces of the channels in the devices remain open and exposed to the environment, which can lead to contamination and loss of samples due to evaporation. This talk will describe recent developments in the fabrication of fully-enclosed devices where the top and bottom faces of the channels are sealed with a layer of toner applied using a conventional laser printer.
Our work is focused on expanding the capabilities of point-of-care diagnostic devices made out of patterned paper by developing simple methods for enclosing the devices, for controlling fluid movement within the devices and for fabricating layered three-dimensional devices. We will demonstrate the fabrication and expanded capabilities of these devices using simple diagnostic assays for glucose and alkaline phosphatase Microfluidic paper-based analytical devices (µPADs) can be considered as the latest, and maybe the most fast-growing technology for the detection of analytes and pathogens at extremely low cost. We present three examples of applications presenting variety of i) designs (lateral flow and microzone plates), ii) chemistry of detection (nanoparticles and dye chemistry), as well as iii) type of analytes (organic compounds, small inorganic anion, and bacteria) and iv) assays (colorimetric, enzymatic, and immunoassays). Microfluidic paper-based analytical devices are a promising technology to develop simple, low-cost, portable, and disposable diagnostic platform for resourcelimited settings. This technology is based in colorimetric bioassays for direct detection of contaminants, metabolites, drugs, diagnostics of disease, etc. Then, the analysis of image is evaluated directly from scanners, or even from cell-phone cameras for telemedicine applications.
We report examples of the fabrication of µPADs in cellulose membrane of Whatman #1 chromatography paper by wax printing, in which we apply for i) detection of adulteration of protein content with melamine and ii) analysis of nitrite in saliva for periodontitis, using a lateral flow µPAD, and iii) immunoenzymatic assays for Toxoplasma gondii detection in pregnant women, using a 96microzone paper plate.
The Derda Lab is interested in simple solution for biochemical assays and for culture of cells and bacteria in limited-resource environment. We also promote the concept of "point-of-care production" for point-of-care diagnostic devices. In this presentation we will demonstrate a functional, portable device for the growth of bacteria that can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen but nonpermeable to water. The devices supply bacteria with oxygen and prevent the evaporation of media for a period over 48 hours. The division time of bacteria in this culture are similar to the rates measured on agar plates and in shaking cultures. The growth of bacteria could be quantified using a flatbed scanner or a cell phone camera. The majority of measurement and fabrication procedures have been replicated by low-skilled personnel (high-school students) in a low-resource environment (high-school classroom). Accordingly, this platform can be used as both an educational tool and as a diagnostic tool in low-resource environments worldwide.

Dr. Marc Dusseiller Dusjags labs, hackteria.org, Switzerland
Dr. Marc R. Dusseiller is a transdisciplinary scholar, lecturer for micro-and nanotechnology (FHNW, University of Applied Sciences and Arts Northwestern Switzerland), cultural facilitator and artist. He works in an integral way to combine science, art and education. He performs DIY (doit-yourself) workshops in lo-fi electronics, hardware hacking, microscopy, music and robotics. He is co-organizing Dock18, Room for Mediacultures, DYI* festival (Zürich, Switzerland), KIBLIX 2011 (Maribor, Slovenia), workshops for artists, schools and children as the president of the Swiss Mechatronic Art Society, SGMK Currently, he is developing means to perform bio-and nanotechnology research and dissemination (Hackteria | Open Source Biological Art) in a DIY fashion in kitchens, ateliers and in developing countries. Abstract:

hackteria.org | Open Source Biological Art, Zürich, Switzerland Email: marc@dusseiller.ch
We will conclude from experiences in the framework of "Hackteria | Open Source Biological Art" and in educational projects with science students, game-designers, artists, hackers and children. During these transdisciplinary collaborations and workshops we have developed a rich online resource of instructions to build DIY (do-it-yourself) laboratory infrastructure using hacked consumer electronics and also novel methods of cultivating creativity across disciplines and public dissemination of Bioand Nanotechnology.

http://hackteria.org/
The Klapperich Laboratory for Appropriate Healthcare Technologies at Boston University is focused on the design and engineering of minimally instrumented, disposable systems that enable low-cost point-of-care molecular diagnostics. We have invented technologies to perform microfluidic sample preparation for bacterial and viral targets from several human body fluids including, urine, blood, stool and nasowash. We are currently working on devices for the detection of infectious diarrhea, influenza, HIV, gonorrhea, chlamydia, MRSA and cancer biomarkers. At this conference, we will present a minimally instrumented, lab free system for preparing, storing and shipping nucleic acids from field sites to central labs.
We are developing portable diagnostic devices for detecting and treating microbial infections in resource-poor settings. These systems do not contain moving parts nor do they require power for their operation. They consist of a degassed layer of polymer that is preloaded with reagents and antimicrobial agents and enables the simultaneous identification of microbial pathogens and their susceptibility to clinical antimicrobial agents. The devices are inexpensive, are small enough to fit in a pocket, and have a shelf life of several months. Importantly, they require only a single step to load and operate, which minimizes the requirement for technical training. The time-to-detection rivals large, expensive clinical instruments that represent the gold-standard in this field.

Dr. Jonas Tegenfeldt Lund University, Sweden
Dr Tegenfeldt is an associate professor in Physics at Lund University and University of Gothenburg in Sweden. His main research focus is on developing micro and nanofluidics tools for fundamental studies and applications with relevance for biomedicine. Having a basic training in Engineering Physics (MSc, Uppsala University) he continued to persue his PhD in Nanotechnology (PhD, Lund University). Later on he spent his postdoc in the labs of Bob Austin and Ted Cox at Princeton University before returning to Sweden to establish his own group. During his career Dr Tegenfeldt has pioneered the use of electrodeless dielectrophoresis, direct visualization of DNA stretched in nanofluidic channels and recently morphological sorting using deterministic lateral displacement. Currently, his group focuses on a wide range of topics including fundamental polymer physics, advanced labeling schemes for DNA, and deformability based sorting and morphology based sorting of cells with special emphasis on pathogen enrichment for diagnosis.

Jonas Tegenfeldt
Division of Solid State Physics, NmC at LU, Lund University, PO Box 118, S-221 00 Lund, Sweden Email: jonas.tegenfeldt@ftf.lth.se Diagnosis of many parasitic disease such as sleeping sickness largely relies on direct observation of the pathogen. For sleeping sickness the relevant parasitemia is however so low that an enrichment is absolutely necessary to be able to find the parasites within reasonable time in a microscope. Using microfluidic sorting devices based on deterministic lateral displacement and made sensitive to the difference in morphology between the erythrocytes and the parasites we have shown proof of principle of extraction of parasites from whole blood. Our technique can be extended so that it is sensitive to the deformability of the cells as well, which in turn may be relevant for extraction of malaria infected erythrocytes. We are developing a microfluidic technology for point of care and patient self-testing of a complete blood count. The miniature cytometer is based on a compact sample processing cartridge with cell analysis chip and reader format. The technology is capable of performing a 3 part differential white blood cell count, platelet count, red blood cell count and haemoglobin measurement using a small sample (uL) High sensitivity and low co-incidence can be achieved without the need for sheath flow or optical scattering techniques that are used in main stream haematology analyzers. Cell counts in whole blood are performed in two parallel channels, one for red blood cells and platelets and another for a three-part differential white blood cell count with an assay time of a few minutes. Concordance tests on clinical samples show excellent correlation against large scale commercial blood analysis equipment. The technology can be further extend for other cell characterization assays such as CD4+ counting for HIV.
The challenge of micro-and nano-fabrication lies in the difficulties and costs associated with patterning at such high resolution. Instead we pattern at the large scale, which is easy and inexpensive, and rely on the heat-induced relaxation of pre-stressed polymer sheets -commodity shrink-wrap film -to achieve our desired structures. Using this approach, we have demonstrated that we can create fully functional and complete microfluidic devices with integrated nanostructures, printed electronics, and even optical components, all within minutes. These devices can be created for only pennies per chip and without any dedicated costly equipment. Because this process is compatible with roll-to-roll plastic processing, it is also scalable and cost-effective enough for point of care applications. We have developed a simple and low-cost microfluidic device for blood plasma separation. In order to detect disease biomarkers from whole blood sample, high-efficiency blood plasma separation is required. Although several types of microfluidic devices have been demonstrated, most approaches rely on high precision microchannels, addition of chemicals and a syringe or vacuum source. We developed a novel microfluidic device that is able to efficiently separate plasma from blood using a simple one-step manual process. The device is low-cost and desirable for low-resource, point-ofcare diagnostics. The "neglected diseases" such as sleeping sickness have a devastating impact on rural communities, in sub-Sahara Africa. The levels of infection within individuals are sufficiently low that point of care instruments must show extra-ordinary sensitivity. We show that low power cell separation methods, based upon electrical and-or acoustic fields, can be combined with mobile phone technologies to enrich and detect micro-organisms at appropriate levels (50 parasites/ml) Resource-constrained settings have limited access to the tools, methods, and expertise required to maximize the earning potential of small-scale, sole-proprietor farms. As a result, proper testing procedures for agricultural goods (e.g., the safety of consumables or the health livestock) are frequently disregarded or falsified. We are developing a suite of low cost, rapid, point-of-care diagnostic assays that are aimed at improving economic outcomes for small shareholder farmers by providing them with tools to better manage their dairy herds and improve the quality of their dairy and maize products. The use of these assays will protect human and animal health and, by assuring the quality of products, increase the incomes of farmers. Aqueous multiphase systems (MuPSs) of polymers and surfactants provide self-assembling stepgradients in density. The interfaces between liquid phases are thermodynamically stable and define a step in density, which can be as small as Δρ = 0.001 g/cm 3 . When a cell sediments through a MuPS under centrifugation, it will collect at an interface between phases that brackets its density. This technique allows blood cells to be rapidly sorted by density and provides important hematological data. The proper choice of densities allows enrichment of cells affected by diseases such as malaria and sickle cell disease.
Density provides a physical marker for cells. Using the unique, self-establishing density steps formed by mixtures of immiscible solutions of aqueous polymers, we are developing tests to do basic hematology tests as well as to detect diseases and disorders that affect cell densities, such as malaria and sickle cell disease.
Dr. Reginald Beer National Security Engineering Division, Center for Micro-and Nanotechnologies, Lawrence Livermore National Laboratory Dr. Reginald Beer is a Medical Diagnostics Initiative Leader who develops Lab-on-Chip technologies for time-critical Molecular Diagnostics applications. His interest in real-time detection is focused on supplying the physician or emergency responder with tools for rapid and accurate diagnostics under harsh conditions. His research and publications in digital PCR, on-chip miniaturization, and rapid amplification have lead to several new technologies for improved diagnostics applications and outcomes. Abstract:

Reginald Beer
Center for Micro-and Nanotechnologies, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550 Email: beer2@llnl.gov The global reach of the U.S. military increases the likelihood of civilian or military personnel to the exposure to emerging infectious diseases and pandemics. Since the 1970s, new emerging infectious diseases have been identified at the unprecedented rate of one or more per year. Many naturally occurring infectious diseases and pathogens are well-suited for weaponization by an adversary for biological warfare. Effective public health systems to counter are essential to mitigating and preventing risks from biological threats. The global health security and biodefense nexus represents an opportunity to define shared objectives for threat reduction activities because the same tools required to prevent, detect, assess, report, and respond to infectious disease outbreaks will increase the ability to counter biological threats whether natural, accidental or deliberate. An unexplained but contained outbreak of anthrax might be traceable to natural causes or deliberate release, either of which would require the same suite of medical countermeasures and defense capabilities. More importantly, the global health security and biodefense nexus offers a clear and compelling rationale for sustained-engagement and cooperation with international partners. Promoting global health security is a shared responsibility that requires global cooperation, sustained-engagement, and enabled by low-cost, deployable technologies.

Ms. Nana Yaa Boadu
Background: Recently adapted World Health Organization (WHO) policies in Ghana recommend testing before treating all suspected malaria patients above five years of age. The original WHO guidelines refer to patients of all ages. Rapid diagnostic tests (RDTs) allow for quick and reliable diagnosis of malaria in peripheral facilities with limited laboratory capacity, without having to rely exclusively on clinical diagnosis. Implementing national health policies in limited-resource settings can be challenging for frontline health services workers. Such challenges may be amplified where implementation involves the uptake of new technology, such as RDTs. Studies reveal variable extents to which test results are followed for guiding fever case management across sub-Saharan Africa. Little is known about health workers' strategies for integrating RDT-use with fever case management in resource-constrained environments. Objectives: 1) to examine national guideline adherence among health workers performing rapid malaria diagnosis; 2) to investigate health workers' strategies for point-of-care malaria testing with RDTs; and 3) to understand how health workers integrate policy with practice amidst limited resources. Methodology/methods: A focused ethnography involving rural/peri-urban district health workers in direct observations, interviews and focus group discussions on rapid diagnostic testing for malaria between April and June 2012. Results: This paper presents early findings on how health workers in this study integrate RDT-use and applied policies for rapid malaria testing in their facilities. Lessons drawn will be useful for informing optimization strategies in the application of RDTs and similar point-of-care diagnostic technologies in developing country health settings.

Aga Khan University Hospital, Nairobi, Kenya
Abstract:

Daniel Maina Aga Khan University Hospital, Nairobi
The AKU hospital committee is tasked with the responsibility of overseeing the formulation and implementation of Point of Care Testing policy within the hospital. Amongst the challenges faced by the committee are issues such as training the end-users on quality assurance, method validation, and the fact that we don't have national guidelines or a regulatory framework to regulate POCT and/or equipment used across various institutions. We describe the development of open channel microfluidic devices and well-plates constructed using ultra-hydrophobic paper as the structural material. The working principle on which these paper-based devices are based is the almost superhydrophobicity generated by covalent grafting of non-polar hydrocarbons on a paper substrate. The elasticity of the paper allows us to emboss different channel patterns on a paper substrate. We can also generate analogous patterns by cutting and stacking the layers of paper and tape. These devices are low-cost and fabrication can be done in a few hours. We discuss the fabrication and application of these paper-based microfluidic devices (see figure below) in crystal growth and fluid flow. We also observe laminar flow with two or three fluids and demonstrate that these devices are analogous to regular PDMS-based devices. These devices can find uses in bio-analytical, diagnostics, genomics, and chemistry laboratories among others.

Makerere University, Kampala, Uganda
Abstract: Molecular diagnostic tests are paramount especially in diagnosis of Trypanosoma brucei gambiense infections where the parasitaemia is usually very low. In this study we compare two simplified molecular tests for the diagnosis; for nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) of clinical samples from patients and serological suspects of T. b. gambiense infection in Uganda. A total of 116 samples were analyzed and the diagnostic sensitivity and specificity of NASBA was 90.6% (95%CI; 80.7-96%) and 100% (95%CI; 94.9-100%), while the sensitivity and specificity for LAMP as compared to microscopy as the reference test was 79.7% (95%CI; 68.2-87.9%) and 100% (95%CI; 91.6-100%) respectively. Both index test showed a highly significant statistical difference in accuracy when compared to microscopy (P=0.0001). Of the suspect samples tested, 4 (13.3%) and 18 (60%) of the samples tested positive with NASBA and LAMP respectively, and 3 of these samples gave a positive result in both NASBA and LAMP. LAMP and NASBA-OC showed comparable diagnostic accuracy when used to diagnose HAT. Larger studies are recommended to explicate the function of these tests in detecting CATT suspect samples and follow-up of such to parasitological conversion of the suspects. Human African trypanosomiasis (HAT) is a vector borne parasitic disease. It is considered a neglected re-emerging disease of public health importance affecting mainly the poor in remote rural African communities. The diagnosis, particularly the stage determination of the disease is critical in the choice of therapeutic intervention. There is need for improvement of diagnostic methods as parasite detection remains insufficiently sensitive and cumbersome. Up to now, a simple serological field applicable diagnostic test for T. b. rhodesiense infection is yet to be implemented. Nanobody® (miniature antibody) technologies are a recent discovery in the field of disease diagnosis. Derived from an in vivo matured camelid immune cell complementary DNA (cDNA), nanobodies have high potential for diagnostic applications in HAT. These recombinant miniature antibodies can be engineered against the conserved regions of variable surface coat antigens on the trypanosome and, conjugated to molecules to increase their sensitivity. Increased investment in malaria research and control leading to improved distribution of insecticidetreated bednets and introduction of artemesinin derivatives in the last two decades has resulted in an overall reduction in the incidence of malaria disease. This has been coupled with a reduction in transmission levels making malaria elimination a possibility. However, complete control and eradication cannot be achieved within a framework of poor diagnostic techniques and policies that tend to ignore low grade parasitaemia especially in asymptomatic carriers that provide a pool for transmission of malaria. The current techniques for malaria diagnosis include microscopic examination of peripheral blood films, antigen detection using rapid diagnostic tests (RDTs) and molecular based methods. RDTs and microscopy may not be reliable especially when parasitaemia fall below 5-10parasites/µl, while molecular methods have a sensitivity and specificity of 100% at 1 parasite/µl but are yet to be developed for point of care use. Microscopy is subjective and heavily relies on the proficiency of the user. Furthermore, there is evidence that RDTs may not be very sensitive especially in varying transmission intensities and ecological settings. We discuss the importance of diagnostic techniques in the fight to eradicate and control malaria and foreseen challenges and possible solutions.

IMPORTANCE OF RAPID AND SENSITIVE DIAGNOSTIC IN ACCURATE ESTIMATION OF HUMAN AFRICAN TRYPANOSOMIASIS MAGNITUDE IN ENDEMIC AREAS IN TANZANIA
Malele, I.I. 1  Sleeping sickness is one of the neglected tropical diseases. More than 60% of the total cases of Human African trypanosomiasis (HAT) reported locally originate from western Tanzania. Cases from Serengeti Ecosystem are reported mostly among tourists. HAT cases, are directly related to the prevalence of human infective trypanosomes in the vectors or host animals. Traditional detection microscopically have short comings as the method doesn't discriminate between human and animal disease causative parasites and is less sensitive. Reliance on such data, tend to underestimate the actual prevalence and thus the risks of HAT. Molecular diagnostic tools were The most widely used tools for diagnosis of S. mansoni, stool examination and serologic detection, are limited by low sensitivity or inability to distinguish between current from former infections, respectively. As a result, there is not an accepted "gold standard" for S. mansoni diagnosis for evaluation of new diagnostic tools. However, recent development of a semi-quantitative PCR that detects schistosome DNA stool provides a tool with increased sensitivity and specificity for this purpose. We utilized the PCR method to help evaluate two versions of a point of contact (POC) test designed to detect circulating cathodic antigen (CCA) in urine of people infected with schistosomes. 1898 school children aged between 8-12 years from Rarieda District of western Kenya took part in the study. Three stool and three urine samples were collected on consecutive days for testing by Kato-Katz and the CCA cassettes. A portion of the first day's stool was preserved in ethanol and subsequently tested for presence of schistosome DNA by PCR.In addition, serum from a single blood sample was tested by ELISA for anti-schistosome IgG. Children who were infected with S. mansoni were treated using praziquantel. Results: Compared to PCR, the initial version of the POC urine test (CCA-M) on any of the 3 days had the highest sensitivity at 92.11%, followed by Kato=-Katz at 70.44%. The alternate version of the POC test (CCA-A) and ELISA had sensitivities of 52.56% and 50.16%, respectively. This results show that PCR is a promising gold standard assay that can be used when evaluating the performance of different tests used for the diagnosis of S.mansoni infection present in Schistosomiasis endemic areas.

University of Notre Dame
The 15-50% prevalence of substandard and counterfeit medicines in developing countries1 unnecessarily increases morbidity and mortality for treatable diseases and favors the emergence of drug resistant disease organisms. 2 We are developing field screening tools for pharmaceuticals based on Paper Analytical Devices (PADs). PADs cost less than $US 0.35 apiece to fabricate, as we will describe at the workshop. They can carry out analytically complex tasks, but they are userfriendly, transportable, do not need electrical power or batteries, and can be automatically read using only a cell phone. Our first PADs test for acetaminophen, artemisinin combination therapy (ACT) drugs, antibiotics and related excipients. Our group is working to expand the library of pharmaceutical analytes using both chemical tests and biochemical methods, such as the use of genetically engineered yeast. We are working to transition this technology from the lab to clinics and will begin field testing in Kenya in June of 2012.

KEMRI-ITROMID
My PhD work involves the development of novel assays for Yellow Fever Virus a zoonotic disease. Hence accorded a chance to attend the workshop will enable me equip myself with the current treads in point of care diagnostics. In resource limited settings the provision of POC aids in averting outbreaks especially in viral heamorrhagic fevers once reliable tests are availed in disease prone zones. The worldwide emergence of arbovirus infections is unparalleled in East Africa where Yellow Fever, Dengue, RFV, Onyong-nyong, Crimean Congo Hemorrhagic Fever and Chikungunya viruses caused disease epidemics in humans in the recent past. The Arbovirus incidence and Diversity (AVID) project lead by icipe aims at strengthening regional capacity for disease surveillance and diagnostics. The project makes use of cutting edge high-throughput diagnostic and sequencing technologies to determine the circulation of arboviruses among human, livestock, wildlife and arthropod vectors in selected RVF epidemic-prone hotspots of Kenya. Samples are screened using family specific primers with MassTag PCR technology, a new technology PCR based on Mass Spectrometric detection of end product. Detection of specific virus is then done using High Resolution Melting. In addition, metagenomic analysis enabled us to assemble full viral genomes and to discover novel emerging pathogens in unexpected hosts. For example we were able to identify pigs as a potential reservoir for Ndumu virus. Available parasitological tests in diagnosis of HAT have limited sensitivity, frequently leading to misdiagnosis of patients with the commonly low parasitaemia. In such cases, minority trypanosomes are shielded by the host red blood cells (RBCs). Yet accurate diagnosis and staging that rely on these tests are crucial in the successful control of HAT. We explored the potential of selective lysis of RBCs for enhancing the parasite detection. Mouse blood spiked with known numbers of trypanosomes was incubated in a home-made ammonium chloride solution or commercially obtained RBC lysis solution. After 5 minutes, the mixtures were centrifuged and the pellets used to prepare wet smears for microscopy. These parasite concentration procedures increased the sensitivity up to 20 times compared to unlysed blood with the same numbers of trypanosomes, which remained motile for several hours in the lysis solutions. This method can be used to concentrate up to 5ml of blood. The reagents require no cold chain making it easy to incorporate this RBC lysis step into the HAT diagnostic algorithm. This could provide template for use with advanced parasite visualization tools, such as the LED fluorescence microscope or molecular diagnostics such as the loop-mediated isothermal amplification (LAMP) of DNA and PCR. Malaria presents one of the major health issues worldwide, with over 80% of all cases occurring in Africa. Several HSPs which are localized in the mitochondria are highly conserved and universally present molecular chaperones that protect cell structures and organelles against thermal, chemical and redox stress. These proteins play crucial roles in folding/unfolding/assembly of proteins, transport/sorting of proteins into correct subcellular compartments, cell cycle control, signaling, and antigen presentation. This is a protective system that is very essential for Plasmodium falciparum survival. Several antimalarials increase the oxidative stress and lead to up regulation of HSPs, therefore combination therapy with small molecular weight inhibitors of HSPs will enhance their effectiveness in killing the parasites or removal by phagocytosis, and In addition several inhibitors of HSPs are currently developed for cancer therapy and might be important as anti-malarials. There are Four major subclasses of HSPS viz; Hsp90, Hsp70, Hsp60, and small Hsps. This study will assess the antimalarial activity of small molecular weight inhibitors of heat shock proteins using three specific objectives: by determining the growth of P falciparum in presence of a combination of small molecular weight inhibitors of HSPs and the commonly used antimalarials (artemisinins, aminoquinolines and antifolates) in vitro, comparing the in vitro activity of antimalarial drugs and small molecular weight inhibitors alone and evaluating the potential of in vitro cross-reactions between the two drugs. Parasite isolates will be cultured using the candle jar technique, drug response assay carried out by subculturing the parasites in the presence of various antimalarial drug concentrations and small molecular weight inhibitors of HSPs, in vitro activity of small molecular weight inhibitors assessed as the drug concentration that inhibits 50% of parasite growth (IC50) using the SYBR green I microtest. Relative levels of HSPs mRNA levels will be measured using qRT-PCR while, relative levels of HSP proteins will be measured using western blots techniques. The non-parametric Kruskal Wallis and Dunn's post test will be used to compare the in vitro activity of antimalarials with that of the small molecular weight inhibitors and Spearman correlation analysis to evaluate the potential of in vitro cross-reaction patterns of the two drugs. Bioinformatics' (NCBI, PlamoDBand Swiss model) databases will be used to check for isolates, 3D structure, drugability, identity, similarity and homology of the HSPs with those of the host. The significance of the study will be the identification of new compounds that target HSPs and are additive to antimalarials that can be assayed invivo to determine their efficacy and for use in the treatment of malaria  Human African trypanosomiasis (HAT) is a vector borne parasitic disease. It is considered a neglected re-emerging disease of public health importance affecting mainly the poor in remote rural African communities. The diagnosis, particularly the stage determination of the disease is critical in the choice of therapeutic intervention. There is need for improvement of diagnostic methods as parasite detection remains insufficiently sensitive and cumbersome. Up to now, a simple serological field applicable diagnostic test for T. b. rhodesiense infection is yet to be implemented. Nanobody® (miniature antibody) technologies are a recent discovery in the field of disease diagnosis. Derived from an in vivo matured camelid immune cell complementary DNA (cDNA), nanobodies have high potential for diagnostic applications in HAT. These recombinant miniature antibodies can be engineered against the conserved regions of variable surface coat antigens on the trypanosome and, conjugated to molecules to increase their sensitivity. Molecular diagnostic tests are paramount especially in diagnosis of Trypanosoma brucei gambiense infections where the parasitaemia is usually very low. In this study we compare two simplified molecular tests for the diagnosis; for nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) of clinical samples from patients and serological suspects of T. b. gambiense infection in Uganda. A total of 116 samples were analyzed and the diagnostic sensitivity and specificity of NASBA was 90.6% (95%CI; 80.7-96%) and 100% (95%CI; 94.9-100%), while the sensitivity and specificity for LAMP as compared to microscopy as the reference test was 79.7% (95%CI; 68.2-87.9%) and 100% (95%CI; 91.6-100%) respectively. Both index test showed a highly significant statistical difference in accuracy when compared to microscopy (P=0.0001). Of the suspect samples tested, 4 (13.3%) and 18 (60%) of the samples tested positive with NASBA and LAMP respectively, and 3 of these samples gave a positive result in both NASBA and LAMP. LAMP and NASBA-OC showed comparable diagnostic accuracy when used to diagnose HAT. Larger studies are recommended to explicate the function of these tests in detecting CATT suspect samples and follow-up of such to parasitological conversion of the suspects. Our research group has developed simple, low-cost, paper-based colorimetric test strips for the detection of water and food contaminants, such as organophosphate pesticides,1 heavy metals,2 and coliforms.3 The test strip utilizes sol-gel based bio-immobilization methods in conjunction with ink-jet printing for the deposition of various reagents onto paper to produce paper-assays.4 The inkjet printing can be performed with a piezo ink-jet printer (Fujifilm Dimatix DMP-2800) or a thermal ink-jet printer (Canon Pixma MP280) and when required a hydrophobic barrier can be created with a wax printer. 5 We will demonstrate the use and fabrication of paper test strips on site. Preliminary results from the 2012 field tests in Kisumu, Kenya will also be discussed.

Daniel Maina Aga Khan University Hospital, Nairobi
The AKU hospital committee is tasked with the responsibility of overseeing the formulation and implementation of Point of Care Testing policy within the hospital. Amongst the challenges faced by the committee are issues such as training the end-users on quality assurance, method validation, and the fact that we don't have national guidelines or a regulatory framework to regulate POCT and/or equipment used across various institutions. Background Human African trypanosomiasis (HAT) or sleeping sickness is a tropical and infectious disease that leads to a complex neuropsychiatric syndrome. It is characterized by two stages, that is, early hemolymphatic stage and late meningoencephalitic stage, primarily based on the detection of white blood cells and/or trypanosomes in the cerebrospinal fluid. The validity of this criterion is, however, debated, and novel laboratory biomarkers are required for accurate staging and subsequent treatment. IL-6 has been shown to play a significant role in neuropathogenesis and as a blood-brain barrier modulator with potential as a parameter for HAT stage determination. Methods Seven adult vervet monkeys (Chlorocebus aethiops) were utilized in this study. Four were experimentally infected with Trypanosoma brucei rhodesiense and treated sub-curatively at 28 days post-infection. Three control animals remained uninfected. Cerebrospinal fluid (CSF) and blood samples were obtained at weekly intervals for 56 days. Sera harvested from blood were used to quantify IL-6 by flow cytometry. Total protein and albumin were quantified in the sera and CSF samples using colorimetric techniques. Results: The serum IL-6 was up-regulated in the transitional stage of infection while CSF IL-6 was up-regulated in the late stage coinciding with relapse parasitaemia. Serum albumin showed a significant decrease (P < 0.05) upon infection only returning to pre-infection levels on sub-curative treatment. Serum total protein significantly increased (P < 0.05) in early stage of infection and only returned to pre-infection levels after subcurative treatment. Cerebrospinal fluid total protein levels showed a significant increase (P < 0.05) in late stage of infection.

University of Nairobi
As a clinician correct diagnosis defines the management outcome of the patient and being in a third world country, Kenya it is almost everyday that one has limited resources to aid in the diagnosis. This has prompted use of simple diagnostic methods which most have been outdated and I would really love this opportunity to get updated and equipped with these skills for better management of my patients. The most widely used tools for diagnosis of S. mansoni, stool examination and serologic detection, are limited by low sensitivity or inability to distinguish between current from former infections, respectively. As a result, there is not an accepted "gold standard" for S. mansoni diagnosis for evaluation of new diagnostic tools. However, recent development of a semi-quantitative PCR that detects schistosome DNA stool provides a tool with increased sensitivity and specificity for this purpose. We utilized the PCR method to help evaluate two versions of a point of contact (POC) test designed to detect circulating cathodic antigen (CCA) in urine of people infected with schistosomes. 1898 school children aged between 8-12 years from Rarieda District of western Kenya took part in the study. Three stool and three urine samples were collected on consecutive days for testing by Kato-Katz and the CCA cassettes. A portion of the first day's stool was preserved in ethanol and subsequently tested for presence of schistosome DNA by PCR.In addition, serum from a single blood sample was tested by ELISA for anti-schistosome IgG. Children who were infected with S. mansoni were treated using praziquantel. Results: Compared to PCR, the initial version of the POC urine test (CCA-M) on any of the 3 days had the highest sensitivity at 92.11%, followed by Kato=-Katz at 70.44%. The alternate version of the POC test (CCA-A) and ELISA had sensitivities of 52.56% and 50.16%, respectively. This results show that PCR is a promising gold standard assay that can be used when evaluating the performance of different tests used for the diagnosis of S.mansoni infection present in Schistosomiasis endemic areas.

Bungoma District Hospital
Glucometer is a patient monitoring and management tool used in measuring blood glucose level. The medical device is commonly used to measure blood glucose level in diabetic patient and on patients on medication: it producers within 6o seconds in addition it uses very little blood from finger tips. Fortunately or unfortunately the device has been manufactured by different companies all over the world this has been a benefit to consumer and also a curse: as result of mass production of glucometers there has been significant variation on results among the medical device. To contain this situation food and drug administration has certified glucometers to be used in blood glucose measurement: however this initiative is still to be adapted by most countries. Glucose level variation more than 1.0mmol/L or more than 10% significantly changes the direction of management of the patient. These changes are critical while handling hypoglycemic or hyperglycemic patient A higher variation will mislead clinicians in managing the patients: as result death may occur due to hypoglycemia ,most diabetic patients will willingly buy this device unaware of its accuracy to produce accurate blood glucose measurement, this posses a great challenges in patient managing himself and herself alone Poster 15: Tsetse flies (Genus Glossina) are the primary vector of trypanosomosis and the only vector capable of transmitting trypanosomes cyclically. This vector is the causative agent for African animal trypanosomosis (Nagana) and Human African trypanosomosis (Sleeping sickness). Tanzania has an area of approximately 937,000 square kilometers and about 60% of the land is infested with seven tsetse species. Trypanosomosis (Nagana) is considered to be one of the most important disease of livestock and is estimated that some 4.4 million livestock are at risk (FAO, 2000), the disease reduces the productivity directly as a consequence of mortality and morbidity and indirectly through its impact on land use and rural development where as farmers normally tends to avoid tsetse infested areas. Mpanda district has 321,000 hectares of land infested by tsetse fly, the areas infested by tsetse fly including the following villages Ugalla, Mnyamasi, Karema, Mpimbwe and Ihumbe. The number of livestock in this district are 219,779(Cattle), 105,063(Goat) and 70,976(Sheep). The presence of tsetse and trypanosomosis in this district cause a big constraint to livestock development through its effect on livestock distribution as the areas which are tsetse infested have reliable source of water and good pastures but livestock keepers tends to avoid this areas and concentrated in small area which is tsetse free and thus cause land degradation due to overgrazing as resulting to environment destruction leading to desertification and poor nutrition to livestock which influence low livestock production items of milk and poor quality meat. Ugalla village is known as endemic foci for Human African trypanosomosis (HAT) so in this village there is a committee for tsetse control and currently deployment of impregnated targets is going on, this project is aiming to integrate different techniques for tsetse and trypanosomosis control. The vision of this project is to reduce trypanosomosis prevalence in livestock by 90% this can be achieved through control of insect vector (tsetse fly).

Lyaruu, Eugen
Malaria in pregnancy is a public health problem in sub-Saharan Africa and represents enormous diagnostic challenges such as nonspecific clinical representation of malaria, lack of effective diagnostic facilities and personnel, and absence of regulatory standards for diagnostic tests. Reproducible animal models are required to overcome these challenges. The human-like structure of the baboon placenta and the cyto-adherent property of Plasmodium knowlesi justify the baboon-P. knowlesi model. In our approach, pregnant and non-pregnant baboons were experimentally infected with P. knowlesi H strain parasites (1x106). They were monitored for parasitaemia, clinical symptoms (from day 2 post infection) and histopathology. Our findings show that parasitaemia levels of the non-pregnant infected (NPI) group was higher (4%) than the pregnant infected (PI) group (3%). The placental parasitaemia was on average over 19 fold higher than the peripheral parasitaemia in the same animal. Histopathological findings confirmed placental parasitaemia and sequestration. Findings in the baboon-P. knowlesi model correlate well with findings observed in humans. Consequently, the baboon-P. knowlesi model of malaria is a promising model of choice that can be used in the development of an effective diagnostic tool for placental malaria.

INSTITUTE OF PRIMATE RESEARCH
Being a scientist and veterinarian I would be interested to learn quick and low cost diagnostics which are essential especially in the African continent. This because of the low high poverty level which limits many people to access good health care for themselves and for their livestock Poster 18:

DIAGNOSTICS EVALUATION AND PERFORMANCE LABORATORY FOR OPERATIONAL YARDSTICKS
Tuan Nguyen, Tom Slezak, Crystal Jaing, Larry Dugan, Reg Beer and George R. Farquar

Lawrence Livermore National Laboratory
Lawrence Livermore National Laboratory (LLNL) is currently developing a new laboratory capability specifically designed to evaluate new and developing diagnostic platforms. The Diagnostics, Evaluation and Performance Laboratory for Operational Yardsticks (DEPLOY) is housed in a set of existing laboratories that have the capability to evaluate the engineering as well as the biology of diagnostic tests. LLNL's capabilities for detector testing and evaluation come from over 20 years of research, development, testing, evaluation and fielding in biological detection and diagnostic testing. The presentation will also highlight three of our ongoing projects in diagnostics (DNA-Microarrays, LAMP and fast PCR). LLNL has developed a DNA Microarray that focuses on vertebrate-infecting pathogens (as opposed to just human-infecting) because effective biosurveillance in Kenya or other developing nations must have the capability to establish baselines and detect deviations for not only humans but also the other hosts, vectors, and reservoirs. LAMP isothermal amplification has shown promise in limited resource settings due to its simplicity.
We have designed, and tested a colorimetric, disposable Point-of-Need (PON) molecular diagnostic that accomplishes sample preparation, isothermal nucleic acid amplification and detection in a single tube in about 1 hour. PCR is the State-of-the-art for molecular diagnostics and detection because of its unmatched specificity and sensitivity. Existing PCR instruments require ~30 minutes (typical of fast systems) to perform their assays. We achieved 30 cycles of amplification in two minutes and eighteen seconds.

This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
Poster 19:

Haramaya University
A cross sectional study was conducted on 906 apparently healthy slaughtered camels and on 120 suspected humans in Ethiopia to investigate the pathology of camel tuberculosis (TB) and characterize its causative agents as well as to assess public health importance of the disease. The prevalence of camel TB was 10.04% (91/906) on the basis of pathology and it was significantly higher in females (x2 = 4.789; P = 0.029). Mycobacterial growth was observed in 34% (31/91) of camels with grossly suspicious TB lesions. Upon further molecular characterization using multiplex PCR, 68% (21/31) of the colonies showed a positive signal for the genus Mycobacterium, of which two were confirmed Mycobacterium bovis by RD4 deletion typing. Further characterization of the two M. bovis at strains level revealed that one of the strains was SB0133 while the other strain was new and had not been reported to the M. bovis database prior to this study. Hence, it has now been reported to the database, and designated as SB1953. On the other hand, 6 of the 8 human isolates subjected to RD9 deletion typing were confirmed to be M. tuberculosis. In conclusion, further genotyping of isolates is useful towards the efforts made to control TB.

Egerton University, 2 KARI-TRC, 3 Laikipia University College
The haematological parameters of sheep (Ovis aries) an experimental model of human African trypanosomiasis were determined. Ten sheep were used ;eight animals were infected with 1× 104 Trypanosoma brucei rhodesiense while two animals were not infected and hence saved as control .Clinical evaluation was done daily while weekly animals of both groups were bled and blood samples examined for packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet counts, Total white blood cell counts and erythrocyte indexes of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration and (MCHC) . Clinical evaluation of the disease in the sheep corresponded closely to that described in human patients. Infected sheep showed an increase in body temperature, MCH and MCHC. However the levels of PCV, Hb, MCV and RBC counts declined drastically on infection. All parameters however recovered to control after treatment. Analysis of data revealed significant changes in all these parameters and erythrocyte indexes in infected sheep when compared with controls. From the changes observed in this study, we can conclude that macrocytic anemia was the major feature of the infected sheep.

IMPORTANCE OF RAPID AND SENSITIVE DIAGNOSTIC IN ACCURATE ESTIMATION OF HUMAN AFRICAN TRYPANOSOMIASIS MAGNITUDE IN ENDEMIC AREAS IN TANZANIA
Malele, I.I. 1  Sleeping sickness is one of the neglected tropical diseases. More than 60% of the total cases of Human African trypanosomiasis (HAT) reported locally originate from western Tanzania. Cases from Serengeti Ecosystem are reported mostly among tourists. HAT cases, are directly related to the prevalence of human infective trypanosomes in the vectors or host animals. Traditional detection microscopically have short comings as the method doesn't discriminate between human and animal disease causative parasites and is less sensitive. Reliance on such data, tend to underestimate the actual prevalence and thus the risks of HAT. Molecular diagnostic tools were

University of Nairobi, Nairobi, Kenya
Over 250 million cases of malaria are reported each year, 1 million of which result in death. Currently, no drugs are universally successful in treating malaria due to widespread drug resistance; new antimalarials from natural products of plant biodiversity are desperately needed. However few data are available on their safety, despite the fact that validation of traditional practices could lead to innovative strategies in malaria control. Out of the 45 organic extracts screened for activity against Artemia salina larvae, 23 (51%) of the crude extracts demonstrated activity at or below 100 µg/ml, and were categorized as having strong cytotoxic activity, whereas 20% (9) of the aqueous extracts demonstrated activity at or below 100 µg/ml and were considered to have strong activity against Artemia salina larvae. In the current study, 95.5% of all the screened organic extracts and 76% of the investigated aqueous extracts demonstrated LC50 values <1000 µg/ml, indicating that these plants could not make safe antimalarial treatments. This calls for dose adjustment amongst the community using the plant extracts for the treatment of malaria and chemical investigation for isolation of bioactive compounds responsible for the observed toxicity Poster 23:

Lund University
While size has been widely used as a parameter in cellular separations, shape and deformability remain largely untapped sources of specificity in preparative and analytical microfluidic devices. Devices that are relatively simple to fabricate and use but that are very sensitive to the size, shape and deformability of cells could be used for a wide range of analytical and preparative processes without recourse to expensive cytometers. Furthermore, labelling of cells is not necessary, helping to keep simplicity and cost at a minimum. Our method is based on Deterministic Lateral Displacement1, a mechanism which causes particles to follow trajectories through arrays of micrometre-sized posts based on size, shape and deformability. We have previously shown how morphological differences can be used to isolate parasites from blood2, a method we hope could find utility in the fight against Human African Trypanosomiasis. We have also shown how the deformability of red blood cells can be measured and used as a separation parameter3, a parameter that is greatly affected by infection by the Malaria parasite. The primary questions that remain to be answered, if these devices are to become useful for Point of Care diagnostics are: How simple yet reliable can the fluidic control be? Do we really need large expensive microscopes to see the results of our analysis? And, can we actually deal with real samples taken in the field?

University of Nairobi
My area of expertise is Physiology of Reproduction. I am currently doing a lot of research through postgraduate students on effects of herbal medicines used by herbal practitioners on the reproductive process. The aim is to prove or disprove the efficacy of such compounds in laboratory animals with a view to moving them forward to formal remedies. This workshop, Point of Care Diagnostic Workshop will be very appropriate especially on clinical aspects of my research.

International Livestock Research Institute, PO Box 30709, 00100 Nairobi, Kenya
Contagious bovine pleuropneumonia (CBPP) is an important cattle disease, which is present in many countries of sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides. CBPP greatly impacts livestock-dependent communities by restricting market access and reduced food supply. The current diagnosis of CBPP relies on tests that have limited sensitivity and are therefore only useful at herd level. The aim of the project was to identify and compare immunogenic Mycoplasma proteins for the development of improved diagnostic assays for CBPP. Immunogenic Mycoplasma proteins have been identified using two-dimentional electrophoresis combined with immunoblot, phase display libraries, as well as Luminex assay. Synthetic versions of Mycoplasma genes have been cloned, expressed and purified and subsequently tested using ELISA. The proteins were screened for their potential as diagnostic antigens using more than 100 well-defined experimental and field sera. Additionally, the presence of the corresponding genes has been verified in a large collection of M. mycoides subsp. mycoides strains.

Johnson Nasimolo
University of Nairobi I am working on the second stage of trypanosomiasis using a mice model, and potential utilization of natural products in the disease. Diagnostic markers are important in determining cure in the mice model. Trypanosoma congolense belongs to the subgenus Nannomonas and is the lead causative agent for African Animal Trypanosomiasis (AAT). T. congolense infections cause losses worth 4.5 billion dollars annually to livestock dependent economies in Sub-Saharan Africa. Thus, trypanocidal drugs are in use. However, parasites resistant to these trypanocides have emerged making the treatment of AAT difficult. Parasite resistance to trypanocides, virulence and infectivity, are variable phenotypes revealed during infection. Consequently genotyping provides a useful link between the genetic component and phenotypic manifestation. In this study the ecology, identity and genetics of trypanosomes circulating between bovine hosts and G. pallidipes in areas surrounding the Shimba hills game reserve, Kenya was considered. Six out of the eleven trypanosome species that are detectable by PCR were found to be present in this area, making the percentage level of diversity 54%. This level of diversity was observed for both cattle hosts and G. pallidipes. This finding will greatly influence future sampling studies in this area, whereby either the vector or host will be used to source trypanosomes. Microsatellite genotyping revealed six diverse genotypes of T. congolense subtype savannah. Interestingly two out of the six genotypes were found in a single fly proboscis.
Poster 28: Incidences of diseases such as HIV, malaria, and tuberculosis (TB) are devastating in developing countries. Poor diagnosis and treatment of diseases in these particular regions is exacerbated due to the lack of resources for purchase or production of diagnostic devices.

DEVELOPMENT OF SENSING PLATFORM FOR TB-SPECIFIC ANTIBODIES USING PAPER-BASED BACTERIA CULTURES AND ENGINEERED BACTERIOPHAGE
To address this issue, we aim to develop a point-of-care diagnostic tool using cost efficient materials that is easy to store and easy to analyze, without depending on costly equipment. Our tool uses engineered bacteriophage to detect biomarkers of disease. The advantages of using bacteriophage as opposed to widely used antibody diagnostic assays are that bacteriophage are simply and easily produced from relatively inexpensive materials, can be stored at ambient conditions, and by the nature of bacteriophage reproduction, produces a strong signal from a single detection event.
The principles of this technique involve taking advantage of the infectivity of phage to produce a signal. The coat protein pIII allows the phage to be infective when intact. However, when pIII is cleaved into two halves, the phage has no infectivity. We aim to cleave and modify pIII such that it will come together again only in the presence of the target biomarker: a circulating antibody against TB antigens. Hence, bacteriophage will infect E. coli to produce a signal only in the presence of model antibody.

National Museums of Kenya
One of the research work focus on population genetics of Plasmodium falciparum and the host. Our study focuses on Erythrocyte Binding protein (EBL-1) and the host Glycophorin B. We are analyzing single nucleotide polymorphisms within study of defining the direction of microevolution of these proteins. We currently are interested in expanding the work to demographic genome wide analysis of the gene isolates from regions within Kenya in relation to global signatures at CEPH Hapmap.

Egerton University, Kenya
The main vector for Plasmodium falciparum in sub-saharan Africa is Anopheles gambiae. P. falciparum is the most virulent human malaria parasite. The difficulties in combating malaria are partly due to rapid resistance emergence by the parasites to the available antimalarial drugs. However, the impact of the drug resistance on the development of Anopheles gambiae is unknown. In this study, the impact on the infection efficiency of the drug resistant parasites to An. gambiae and the vector survival rate after successful infected blood meal will be studied. Two hundred laboratory adapted An. gambiae s.s. mosquitoes will be fed on gametocytes of cultured Sulfadoxine pyrimethamine (SP) resistant laboratory strain of P. falciparum (Dd2) via an artificial membrane feeding system. Fully engorged mosquitoes will be maintained on 10% glucose solution in an insectary at relative humidity of 75-80%, and a temperature of 25-27oC and mortality rate monitored twice daily. To estimate the parasite load on the dead mosquitoes, the mosquitoes will be dissected and their head, thorax and abdomen analyzed using circumsporozoite protein ELISA. These data will be compared with those obtained from mosquitoes fed on a SP sensitive laboratory P. falciparum (M24) strain. It is expected that the longevity of the mosquitoes infected with drug resistant strain will be significantly different compared to the mosquitoes infected with the sensitive P. falciparum. This will be an indication that drug resistant P. falciparum strains also changes in virulence in addition to its mutation associated with drug pressure evasion. This study will shed light on P. falciparum virulence vis-a-vis drug resistance. Understanding how the parasite resistance impacts on the longevity of the vector may also aid in malaria transmission control strategies. In addition, a masters degree and a publication of this work on a peer reviewed journal will be achieved.

Kirtika Patel
Moi University, Kenya I am a lecturer and investigator in Immunology in the School of Medicine at Moi University, Eldoret, Kenya. I have been involved in initiating the setting of Immunohistochemistry (IHC) Laboratory, a first of its kind in Western Kenya. Have worked on mutational and risk factor analysis for esophageal cancer. Currently, working on evaluation of hormone-receptor status and growth factor receptor expression as predictive factors for breast cancer prognosis, using FACS and IHC for diagnosing lymphomas and Kaposis Sarcoma, PCR for HPV detection in cervical cancer and molecular analysis of ethnic variation and molecular fingerprint in Wilms' tumor. Other goal is to establish a tissue repository. I am hoping to develop easier ways for diagnosing cancer in a limited resource setting like ours. This workshop will expose me to various research ideas and help in setting up of cheaper and easier way to test for cancer and thus help in delivering excellent point of care. Our research in on African swine fever, a highly fatal devastating disease of pigs. Trying to find out the most common strains present in the country and develop antigen and antibody based diagnostic tools for the disease. Specific viral protein and recombinant protein will be produced and used in the development of the diagnostic technologies. The title of the research work is "Establishment of diagnostic technologies for increased animal production in Uganda. Increased investment in malaria research and control leading to improved distribution of insecticidetreated bednets and introduction of artemesinin derivatives in the last two decades has resulted in an overall reduction in the incidence of malaria disease. This has been coupled with a reduction in transmission levels making malaria elimination a possibility. However, complete control and eradication cannot be achieved within a framework of poor diagnostic techniques and policies that tend to ignore low grade parasitaemia especially in asymptomatic carriers that provide a pool for transmission of malaria. The current techniques for malaria diagnosis include microscopic examination of peripheral blood films, antigen detection using rapid diagnostic tests (RDTs) and molecular based methods. RDTs and microscopy may not be reliable especially when parasitaemia fall below 5-10parasites/µl, while molecular methods have a sensitivity and specificity of 100% at 1 parasite/µl but are yet to be developed for point of care use. Microscopy is subjective and heavily relies on the proficiency of the user. Furthermore, there is evidence that RDTs may not be very sensitive especially in varying transmission intensities and ecological settings. We discuss the importance of diagnostic techniques in the fight to eradicate and control malaria and foreseen challenges and possible solutions.

INSTITUTE OF PRIMATE RESEARCH, JKUAT
My PhD research involves studies on prevalence of toxoplasmosis. Toxoplasmosis affects all warmblooded hosts, including humans. Toxoplasmosis is a leading cause of infectious reproductive failure in humans and animals, and severe disease in congenitally infected children and immunocompromised people. Despite its importance, information on the occurrence and control of this zoonosis in Kenya is limited. The project aims to determine burden of toxoplasmosis and identify T. gondii genotypes infecting humans in Thika District, Kenya. Study population being surveyed includes livestock keepers, slaughterhouse workers, pregnant women, HIV-positive patients and individuals with visual and mental handicaps. The tests being used in the study includes ELISA, PCR, LAMP and sequencing. Trypanosomiasis, is economically important cattle disease in Tanzania inflicting annual losses of 364 million US$ through lowered productivity. The disease problem is propagated by lack of sensitive techniques for diagnosis in most of the affected rural areas. The limitations of available parasitological and serological methods for diagnosis necessitate employment of sensitive and specific DNA-based diagnostic methods to facilitate epidemiological studies of trypanosomiasis. This research project aims at validating the developed primers in the previous study or design new ones for the detection of the three trypanosomes species (Trypanosoma congolense, T.vivax and T.b.brucei) causing African Animal Trypanosomiasis that are circulating in Tanzania. The validation will be done in phases by testing the sensitivity of the Loop Mediated Isothermal Amplification (LAMP) to monitor the efficacy of the treatment in experimental animals. Animals (small ruminants and young cattle) will be cleared and infected then monitored using LAMP and parasitological technique for comparison for four and two week's respectively. Phase three will involve the validation of the technique at large scale. Blood samples will be collected randomly from livestock in the areas that are highly infested with Trypanosome species in Tanzania. After successful validation the technique will be made available to the veterinary department for implementation.

Egerton University, Kenya
Entamoeba histolytica causes human amoebiasis and is among the most common parasitic infections worldwide infecting over 50 million people. Clinical manifestation is due to existence of two morphologically identical species but with different biochemical and genetic makeup. As such, it is of clinical importance to differentiate the two species (commensal and pathogenic), for treatment decision and public health interest. Infection occurs by ingestion of viable cysts from contaminated material such as water and food. Treatment is administered to all diagnosed cases of E. histolytica irrespective whether one has symptoms to avoid the risk of invasion, whereas cases found to involve only E.dispar should not be treated. Correct diagnosis, depends mainly on stool microscopy, which is slow and has low sensitivity and specificity thus cannot be relied upon to distinguish between the two due to its reliance on morphological characteristics. The current study was designed to differentiate E.histolytica and E.dispar by multiplex polymerase reaction in stool samples. The findings of this study are expected to highlight the degree of variation of sensitivity between the two methods and also determine the occurrence of the strains. This will form the basis for further research and management of the complex.

INSTITUTE OF PRIMATE RESEARCH, Kenya
I am involved in the development of an Olive baboon model for Alzheimer's disease. The project entails occlusion of extra-cranial vessels to induce cerebral ischemia. Whole brain is subsequently harvested and pathological indices of Ad equivalence are assessed for with a view to mimic as closely as possible actual AD pathology. White matter lesions ar also examined to generate a template for vascular dementia staging. A major variable is the duration of occlusion and this has been shown to be a determinant in the extent and pattern of white matter pathology, glial cell proliferation and neuronal loss in the grey matter. The ultimate aim is to produce an animal model with the potential for testing and validating candidate therapeutic agents against degenerative diseases of the central nervous system.
Background: Recently adapted World Health Organization (WHO) policies in Ghana recommend testing before treating all suspected malaria patients above five years of age. The original WHO guidelines refer to patients of all ages. Rapid diagnostic tests (RDTs) allow for quick and reliable diagnosis of malaria in peripheral facilities with limited laboratory capacity, without having to rely exclusively on clinical diagnosis. Implementing national health policies in limited-resource settings can be challenging for frontline health services workers. Such challenges may be amplified where implementation involves the uptake of new technology, such as RDTs. Studies reveal variable extents to which test results are followed for guiding fever case management across sub-Saharan Africa. Little is known about health workers' strategies for integrating RDT-use with fever case management in resource-constrained environments. Objectives: 1) to examine national guideline adherence among health workers performing rapid malaria diagnosis; 2) to investigate health workers' strategies for point-of-care malaria testing with RDTs; and 3) to understand how health workers integrate policy with practice amidst limited resources. Methodology/methods: A focused ethnography involving rural/peri-urban district health workers in direct observations, interviews and focus group discussions on rapid diagnostic testing for malaria between April and June 2012. Results: This paper presents early findings on how health workers in this study integrate RDT-use and applied policies for rapid malaria testing in their facilities. Lessons drawn will be useful for informing optimization strategies in the application of RDTs and similar point-of-care diagnostic technologies in developing country health settings. Human African trypanosomiasis is associated with metabolic changes and immunological changes which have not been well characterized. Four Chlorocebus aethiops were experimentally infected with T. b. rhodesiense and late stage disease induced at 28 days post infection (dpi). Ear prick blood for glucose determination and blood samples were obtained at weekly intervals for 63 days. Analysis was done using dry chemistry analysis. In early infection, there was a significant increase in creatine kinase, while during early and transitional stage of infection there was a significant decrease in blood glucose and high density lipoprotein (HDL) and an increase in triglyceride levels.
In the advanced late stage, there was a significant increase in both total cholesterol and low density lipoprotein (LDL) levels. Further investigations should focus on levels of total cholesterol during the follow-up period in curatively treated vervet monkeys. Apart from their importance in disease staging, the changes in lipids levels may also affect the pharmacokinetics of some trypanocides.

Nicholas Kiulia, INSTITUTE OF PRIMATE RESEARCH, Kenya
Background: Group A rotaviruses are a major cause of acute gastroenteritis and severe dehydrating diarrhoea in children under five years of age. The World Health Organization has recommended that rotavirus vaccines be included in all national immunization programs as part of a strategy to control rotavirus-associated diarrhoeal diseases. Hospital -based surveillance of severe rotavirus diarrhoea is therefore crucial in monitoring the impact pre and post-vaccine introduction and also to document changes in genotype distribution. Objectives: To determine the molecular epidemiology of circulating rotavirus strains in the Eastern region of Kenya prior to introduction and implementation of rotavirus vaccination into the national immunization programmes. Study design: During the period September 2009 through August 2011, 500 stool samples were collected from children < 5 years of age admitted for acute diarrhoea in hospitals in the Eastern region of Kenya, and analysed for the presence of group A rotavirus using an enzyme immunoassay. G and P genotypes of these rotaviruses were determined using nested reversetranscriptase polymerase chain reaction. Results: One hundred and eighty nine (38%) of the samples analysed were positive for rotavirus. The following G types were detected: G9 (64%), G8 (13%), G12 (8%), G1 (4%), and G2 (1%). Mixed G types were also detected in 2 samples (2%), and 9 samples (8%) were non-typeable. The following P types were detected: P [8] (47%), P [4] (17%), and P [6] (7%). A mixed P type was found in 11% of samples, and 19% were non-typeable. The most dominant strain was G9P [8] (40%), followed by G8P [4] (10%) and G12P [6] (5%). Other G and P type combinations detected were G1P [8] (4%), G12P [4] (4%), G9P [6] (3%), G9P [4] (2%), G8P [8] (2%), and G2P [4] (1%). Conclusions: The present study divulges the recurring changing genotypes of rotavirus circulating in Kenya with genotypes G9 and G8 being the dominant rotavirus strains circulating in the Eastern region of Kenya between 2009 and 2011. Additionally, the emerging, uncommon G12 genotype was detected for the first time in Kenya.

Hawassa University, Ethiopia
My medical practice involves evaluating, diagnosing and caring for patients of varying medical illness coming from the southern part of the country. Due to lack of standard laboratory and other diagnosing investigations, most of my patients get subjected to empiric therapy which involves administration of multiple drugs with considerable side effects and interaction. An opportunity to upscale my knowledge and skill regarding point of care diagnostic investigations will go a long way in improving the quality of possible care I can deliver to my patients. In addition, my work as a lecturer entails providing clinical training to health officer students who, after graduation, will practice medicine in the most resource constrained parts of the country. The skill from the workshop will also improve the standard of the education I will provide to my students.

INSTITUTE OF PRIMATE RESEARCH, Kenya
I am currently involved in malaria research in respect to developing the non human primate as human model for malaria in pregnancy and cerebral involvement. Determining host parasite interactions, parasite biology, malaria and helminthes coinfection in respect to parasite load and immunological response interactions and preclinical evaluation of blood stage candidate vaccines. Also testing the anti-plasmodial and therapeutic activity of selected medicinal herbs and old drugs as a cheap remedy to malaria. In malaria in pregnancy and cerebral involvement we have a keen interest in identifying molecules that can be used as prototype diagnostic target which are non invasive in human diseases. Demonstration of schistosome eggs in stool/urine is the gold standard for clinical examination of schistosomiasis. These tests have low sensitivity making infections undetectable. Antibody based assays cannot distinguish between past and active infections thus unsuitable for follow up after drug administration. Molecular techniques have high specificity and sensitivity but are expensive and unavailable at the point of care centers. Bioinformatics and Proteomics approaches can be used to identify and characterize schistosome proteins. These proteins originate from parasite life cycle stages and released into the bloodstream and/or urine, derived from worm gut, worm tegument, egg secretions or released products of dead eggs thus good diagnostic targets. Three schistosome proteins were identified, Cathepsin B, Asaparaginyl endopeptidase) and Sm200. Using these approaches, peptide sequences from these proteins synthesized as multiple antigenic peptides were used to immunize rats. Serum from immunized rats were used to test the suitability of these targets using Enzyme Assays, Blot Assays and Immunocytochemistry with worm sections. Results show that multiple antigenic peptides raised antibodies in rats making these proteins possible diagnostic targets that can be developed further to assess their sensitivity aiming at coming up with an assay capable of detecting the lowest number of worms in the host.

Ministry of Medical Services 2 Medical Services -GOK 3 EGPAF-Kisumu
Western Province External Quality Assessment Scheme (WEPEQAS) is performed in the Western Province of Kenya. The scheme is a project that is run by government of Kenya medical laboratory staff supported by APHIA-Plus. The scheme involves results comparison of CD4 and hemoglobin (HB) across machines in the province. The objective of the scheme is to ensure there are accurate and reliable results produced by the CD4 and HB machines in the province. Proficiency test panels are prepared in the provincial headquarters and sent to 17 laboratories in the province. The results are then sent back and analysis is done. The comparison and scoring are done by the use of Mean; Standard deviations and coefficient of variations. There is relatively little variation in regard to different machines platforms in performance. Since 2009, the 17 CD4 and HB machines assessed proved that. However a 5 percent variation should be allowed. Use of point-of-care machines serves the same results as high output and big on-the-bench machines. The former however stands better chance for patient access to services.

MANUALLY ACTUATED MICROFLUIDIC DEVICE FOR BLOOD PLASMA SEPARATION
Paul Scanlan, Farid Amalou and Wenmiao Shu*

School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom
We have developed a simple and low-cost microfluidic device for blood plasma separation that can be operated simply by using the finger. In order to detect disease biomarkers from a whole blood sample, high-efficiency blood plasma separation is required.
Conventional methods such as centrifugal and filtration can be expensive and time consuming, which has limited its usages in the field, at home or for resource limited applications. This has led to the development of microscale separation techniques [1], some based on the afore-mentioned principles [2][3][4] and others in microfluidics. Although several types of microfluidic device have been demonstrated, most approaches rely on high precision microchannels the addition of chemicals or a driving force from an external syringe or vacuum retreatment [5][6][7][8][9]. We developed a novel device that is able to efficiently separate plasma from blood using a simple, one-step, manual process, incorporated into a single chip. The device is low-cost and desirable for low-resource, point-of-care diagnostics.
The chip works using the fact that red blood cells carry a greater density than that of white blood cells and the plasma which make up a whole blood sample [10]. As blood flows through the chip, the effects of gravity draw red blood cells to the bottom of a series of trenches, separating them from the rest of the sample. In order for this to work effectively, it is vital to regulate the rate of flow of blood through the chip [11] and our design uses an applied external pressure over an elastic membrane to create the driving pressure.
In our practical demonstration we will show how the 3-dimensional chip is fabricated and assembled from plastic sheets and adhesive transfer tape. The design of each individual layer can be created using a low-cost CO2 high power laser. The simple fabrication process allows the creation of complex 3-D multi-layer structures where each layer defines the inlet/outlet chambers, microchannels and separation trenches.
Following assembly of the chip, we will go on to demonstrate it in operation by using it to separate a whole sample of animal blood. Similar tests have already been carried out to determine the efficiency of the device, the results of which can be seen in the diagrams in Figure 1. To the visible eye, there is a clear separation between the red blood cells (from sheep) trapped in the separation trench and the clear plasma found in the outlet chambers. Under microscope it is possible to see almost no residual cellular traces found in the extracted plasma, indicating a blood separation efficiency close to 100%.  Background: Malaria kills close to 1 million people annually, mostly young children. Sensitive, affordable, and rapid diagnosis is critical for patient care and to support eradication strategies. Our objective is to modify a standard haematocrit capillary to enable direct testing for malaria by qPCR and melt curve analysis.

Methods:
Reagents for malaria real-time PCR are combined with an acrylamide solution within a capillary to form a 'hydrogel'. The hydrogel is desiccated for long-term storage and to enable sample delivery by capillary action. The gel is rehydrated by dipping the capillary in a 1:5 dilution of a clinical sample. Capillaries are run on a real-time PCR instrument that was developed in our lab.

Results:
The capillary-based assay detects the four major species of Plasmodium directly from unprocessed clinical samples. The limit of detection is 10 parasites/µL of blood. Positive and negative controls were designed to provide quality control for molecular testing in capillaries. We have also shown that hydrogel capillaries can be stored at room temperature for at least 4 weeks with no loss in performance.

Conclusions:
Our innovative platform combines sample collection and testing within a single, lowcost, disposable capillary for molecular diagnostics at the point-of-care. Blood can be added to capillaries directly, without requiring sample processing. Capillaries are also adapted for long-term storage in the field obviating the need for a cold chain to preserve molecular reagents. The sensitivity, affordability and user-friendliness of this technology make it an excellent platform for a variety of clinical applications within resource-limited settings. In human schistosomiasis infections, it is impossible to directly determine worm burden and hence surrogates must be used. In this study we looked at the relationship between worm burden and circulating anodic antigen (CAA) concentrations in serum of baboons infected with S. mansoni . Four groups of baboons (n=2) were infected with 5, 25, 100 and 500 S. mansoni cercariae. 10 weeks post infection, the baboons were perfused and worm burden quantified. Faecal egg output was determined by the Kato Katz technique and microscopy and serum was collected for antibody and CAA determination. CAA levels were measured with the UCP-CAA LF assay that uses the principle of lateral flow through a nitrocellulose strip on which the presence of CAA in a sample is shown by the capture with anti-CAA monoclonal antibodies and visualization with anti-CAA Ab-UCP conjugates. The immobilized UCP is shown by scanning the the strips with a portable lightweight UCP reader. The lower level of detection using 20 µl serum was 30 pg CAA/ml, and 1 pg/ml with 500 µl serum The latter approach involved concentration of the TCA-treated sample and allowed demonstration of an infection with 1 worm per baboon. Also a clear correlation of CAA levels with worm numbers was shown. In addition, using the same techniques, detection of CAA in urine from humans was shown to be highly sensitive and specific, both for S. haematobium and S. mansoni infections. With concentration of 4 ml of TCA-treated urine, even 100% sensitivity was shown in samples with very low infection levels as measured by multilple egg counts and PCR. Also the CAA levels in urine correlated significantly with S.h. and S.m. egg loads.

JKUAT-INTROMID
My studies involve the development of monoclonal antibodies for use in diagnosis of Rift Valley Fever Virus, and my future plan is to avail this diagnosis assay at a point of care. Attending the workshop will grant me an opportunity to interact with scientists who are experienced in this field and I will gained techniques that will be applicable and of great importance to my study.

Samuel Mwangi Njoroge
Jomo Kenyatta University of Agriculture and Technology/ KEMRI Diarrhoea in children under five years, continue to be a major public concern in developing countries like Kenya. Malnutrition can predispose a child to diarrhoea or be a result of persistent diarrhoea. The study design will be cross-sectional in nature and will be nested in a bigger cotrimoxazole prophylaxis clinical trial. The study site is Mbagathi District Hospital and will target the urban-poor population from Kibera. Study sample size will be 217 rectal swabs/ stool samples from severely malnourished children with < -3.0 standard deviation from mid upper arm circumference. The study seeks to identify etiologic agents of diarrhoea and equally important, their drug resistance patterns towards amino-penicillins cephalosporins and sulfonamide. Rectal swabs/Stool will be cultured on MacConkey and Salmonella Shigella Agar. Lactose fermenters colonies will also be screened for extended beta-lactamases, sulfomnamide, and fluroquinolone resistance using a 16 drug panel. Resistant isolates will be screened for extended beta-lactamase genes, dehydrofolate reductase genes or fluoroqinolone resistance genes using qualitative multiplexPCR. Confirmatory test will be done by nucleotide sequencing and comparison made with strains on GeneBank. The project is estimated to cost 1062850 Kenyan shillings with a nine month time-frame. Supervisors will be involved in the management of resources. Findings will be published. Supervisors are Dr. John Kiiru and Dr. Gideon Kikuvi all who have published some work on antimicrobial drug resistance. Dr John is a scientist at KEMRI-center for microbiology research while Dr Gideon is a lecturer/ Deputy Director JKUAT/ITROMID. Sleeping sickness (SS) is a fatal disease caused by a protozoa of the Trypanosoma brucei subgroup.1 The tsetse fly vector injects the parasite while feeding on blood. Initially the parasites proliferate in the hemolymphatic system of infected patients and then spread to the central nervous system (CNS) where they cause increasing neurological dysfunction. Treatment of SS includes a multitude of side effects. In addition, if the parasite is given enough time to invade the CNS, treatment relies to a great extent on Melarsoprol, a highly toxic arsenic derivate with a mortality rate of 5%. This emphasizes the need for a method of early detection with a minimum level of false positives. Accurate detection requires finding the actual parasite, most often done by simple microscopic examination of blood smears, a very time-consuming method with a low detection limit. Here, a key bottleneck is the low concentration of the parasite. Current alternative methods are expensive and inadequate for use in the field.2 Our method provides a dramatically simpler and cheaper alternative with the potential of enabling a fast and cheap point-of-care device with significant impact on the diagnosis of sleeping sickness. Our microfluidic devices rely on Deterministic Lateral Displacement (DLD)3 which our group has shown can be employed to separate particles based on shape4 and deformability5. In the present work we report a simple DLD device, driven simply by hand with a syringe, with which we can analyze several microlitres per minute, resulting in a time per test of ~10 min.

Swaleh Sauda
Kenyatta University I undertake research in natural product chemistry as well as applied analytical chemistry.
• Isolation, biological investigation and structural elucidation of the secondary metabolites from Kenyan medicinal plants that exhibit antimicrobial and antifungal activities, cytotoxicity effects and wound healing properties.
• Investigation of Natural products with antimalarial, trypanocidal and leishmanicidal activities and efficacy of medicinal plants • Chemical profile analysis of Essential oils and determination of their antimicrobial activities.
• Bioavalaibility of Vitamin A, E and C in people living with AIDS ad effects of indigenous foods in the management of HIV and AIDS.
• Quantification of phytochemicals with antioxidant properties from variety of fruits found in selected areas of Kenya.
• Cyanogenic potential levels of cassava cultivars from different Ago-ecological zones in Kenya.

Tendai Gadzikwa
University of Zimbabwe, Zimbabwe I have just recently joined the Chemistry Department at the University, and I am in the process of developing a research program that is feasible with our limited resources. As I am new to the area of diagnostics, I believe that the Point-of-Care Diagnostic Workshop will be an invaluable opportunity for me to learn from those with some experience in the field already. I also hope to develop relationships with other African scientists, and perhaps initiate regional collaborations with them. I am embarking on a collaboration with the Gibbs-Davis group at the University of Alberta. They have developed a DNA detection method based on DNA amplification, which they intend to apply to the detection of TB. My role in this partnership will be to develop a protocol for this method; one that an be implemented easily in labs and clinics in Africa. Dr. Julianne Gibbs-Davis will be demonstrating the amplification/detection method at the workshop.

Sidai Africa Ltd
Sidai Africa Ltd is a newly established social enterprise in Kenya that seeks to offer quality veterinary services and products to livestock keepers, especially the small scale and marginalized ones. A major challenge of offering this service is the unavailability of simple penside diagnostic techniques which would go a long way in improving the quality service we desire. I am particularly interested in simple technologies that can be used in testing for fake drugs that are rife in Kenya especially in the veterinary sector which hardly gets any support from regulatory authorities. African trypanosomiasis is a fatal infection of man and animals. It is often diagnosed as early and late stages based on clinical presentation. Infection with African trypanosomiasis invokes both innate and adaptive immune responses from the host. Macrophages elicited from trypanosomeinfected hosts exhibit increased expression of pro-inflammatory and immune-regulatory molecules with the initial inflammatory response being beneficial to the host at early stages of infection, however, a sustained inflammation can cause pathology. Limitation of aberrant immune responses and maintaining tolerance toward self tissues is required and is the function of Regulatory T cells (Tregs). Although Tregs have been implicated in infectious diseases, particularly in chronic or persistent infections where they may prevent immunopathology, but also compromise pathogen eradication through manipulating antigen presenting dendritic cells (DC), and thus reducing longlasting interactions between effector T cells and DC, induction and mode of action of the various distinct Treg subsets remain ill defined particularly in Trypanosomiasis. This study hypothesizes that T-regulatory (Tregs) cells do not play a functional role if at all expressed, in the severity and clinical course of rhodesiense type of trypanosomiasis in mice. The study will determine and profile expression of pro-inflammatory and T-regulatory cells in the course of trypanosomiasis and to determine the effect of T-regulatory cell depletion on the pathogenesis of trypanosomiasis in Swiss white mice. A Trypanosoma brucei rhodesiense strain from KARI-TRC trypanosome bank will be transfected with a construct containing the gene for green fluorescent protein (GFP) under the control of the metacyclin promoter and the GFP prepared inoculums of 4 x 103 µl-1 be introduced into Swiss white mice. Fluorescent microscopy will be used to identify the parasitemia levels in peripheral blood at intervals of 72 hours four days post infection and in contrast immune cells expressed four days post infection, here-in considered as early stage will be sampled from blood.
The late stage will sample from both brain and blood tissues to be analyzed by flow Cytometry and results compared with the control. This study hopes to identify novel biomarkers associated with progression of trypanosomiasis which should bolster existing diagnostic approaches and should enhance accuracy in detection for effective treatment of the disease. Findings from this study should offer opportunities for identification of possible vaccine targets or identify options for immunotherapy. Trypanosoma brucei is a tsetse transmitted flagellated parasitic protozoon that causes African trypanosomiasis, a disease affecting man and animals. Two subspecies of T. brucei, T. b. rhodesiense and T. b. gambiense cause acute and chronic forms of Human African trypanosomiasis (HAT) respectively. In mammalian hosts, T. brucei evades immune clearance by antigenic variation, the sequential expression of antigenically distinct glycosylphosphatidylinositol (GPI)-anchored variable surface coats called variable surface glycoprotein (VSG). The variable antigen types (VATs) in distinct VSGs are expressed in a reversible and hierarchical fashion, hence are potential candidates for diagnosis. Here, using monkey model, VATs expressed early on infection with T. b. rhodesiense were determined and recombinant proteins expressed and purified using bacterial expression system. Preliminary screening show that a set of these VATs is recognized by infected human serum samples from East African region, and are present in the VSG gene repertoire. Therefore, these VATs are potential candidates for development of a serodiagnostic kit.

Strathmore University
My research is on population genetics of host and parasite genes involved in invasion of malaria. Strathmore University, has recently opened a medical clinic and is interested in becoming a lead player in providing adequate and excellent medical care in the region. The study compared two sets of bioassays designed to evaluate repellency of essential oil of Tagetes minuta against climbing response behaviour of adult, Rhipicephalus appendiculatus, the vector of deadly livestock disease, East Coast fever. The study aimed at evaluating the appropriate bioassay set up suitable for screening repellent essential oils that may become applicable in preventive measures for managing arthropod vectors and vector-borne diseases. All bioassays were conducted under the same laboratory conditions and time. In both bioassays, repellency was dose-dependent and significance differences between doses remained the same at P<0.0001, (n = 5). However, for the same doses, mean percent repellence was lower in the no-choice bioassay (ranging from 17.88±2.39% to 96.5±1.88%) than in the dual-choice bioassay (ranging from 57.92±7.11% to 100.00%). This difference was significant (t (0.05) = 3.256; P = 0.047) but its underlying mechanism however, remained unknown. Probit analysis showed that to achieve the same repellent effect, a higher repellent dose is required in no-choice bioassay than in a dualchoice bioassay, hence the former proving unsuitable for screening purposes. These choice bioassays provide baseline data against which novel tick repellents/attractants may be evaluated for development into agents suitable for providing prophylactic measures in integrated pest management.