Multiplex paper-based designs for point-of-care (POC) diagnostics

Background: Accurate and timely diagnosis is usually the first step towards appropriate disease management. In resource-limited settings, healthcare workers lack proper facilities to perform vital tests, and the diagnosis of disease is often determined by non-specific, physiological symptoms alone. Simple and rapid tests are needed as prerequisite tools for patient care and must conform to the criteria set out by the World Health Organization. To address the challenge of specific diagnosis for diseases that present with similar symptoms, multiplex diagnostic platforms must be designed.Methods: Simple designs for multiplexed paper-based diagnostic platforms were developed. Laser cutting and wax printing were used to create specific patterns on paper to guide the flow of sample and reagents towards reaction zones. Multi-arm and single strip multiplex platforms were designed and tested using Human Immunoglobulin G, Hepatitis B virus surface antigen (HBsAg), and Helicobacter pylori antigens.Results: All designs produced promising results with the lowest limit of detection for antigens being 30ng/ml for single strip designs.Conclusions: The use of glass fibre conjugate pad was found to be more sensitive compared wax-printed chromatography paper. Results from this study indicate great potential for further application in development of diagnostic low-cost paper-based diagnostic devices

Immunochromatographic thread-based test platform for diagnosis of infectious diseases

This research article published by Springer Nature Switzerland AG., 2018Patterning is an important step in fabrication of multiplexed microfluidic devices. Various approaches including cutting, photolithography, wax-printing, plotting and etching have been developed and tested. Recently, using threads has emerged as a convenient and low-cost approach for fabrication of microfluidic devices. We explored the application of threads in combination with nitrocellulose membrane to fabricate multi-channel immunochromatographic diagnostic devices. Microfluidic channels were made using hydrophilic threads and nitrocellulose membrane strips. Household sewing needle was used to weave hydrophilic thread into desired patterns through a double-sided mounting tape. Glass fibre discs were used as conjugate pads while nitrocellulose membrane was used for immobilisation of capture antibodies. Patterned threads were linked to nitrocellulose membrane strips by overlapping so that reagents flowing through threads were eventually transferred to the membrane. The design was tested using IgG, H. pylori and Hepatitis B surface antigen. Continuous flow was observed from hydrophilic threads to the nitrocellulose membrane, and a positive signal was visualised on the membrane within 5 min of sample application. The observed limit of detection ranged between 30 and 300 ng/ml for H. pylori and Hepatitis B, respectively. Using thread and tape offers a promising alternative for patterning of simple, low-cost multiplexed microfluidic diagnostic devices with potential point-of-care applications in resource-limited settings

Seroprevalence of foot and mouth disease in the wildlife-livestock interface and non-interface areas in Tanzania

Abstract A cross sectional study was conducted in the Serengeti ecosystem (wildlife-livestock interface) and central part of Tanzania (non-interface) area to determine the prevalence of foot and mouth disease (FMD) in Serengeti, Bunda, Kongwa and Iramba Districts. Seroprevalence investigation using 3ABC-ELISA technique indicated that the overall prevalence of antibodies against FMD virus was 66.3%. Significantly high prevalence was recorded in wildlifelivestock interface areas (71.5%) compared to non-interface areas (61.0%). District-wise, higher prevalence was recorded in Kongwa district (89.0%) followed by Serengeti (78.0%), Bunda (65.0%) and Iramba (33.0%). Specieswise, higher prevalence was found in bovines (69.8%), ovines (52.4%) and caprines (11.1%). From various risk factors, ecosystem distribution (X 2 = 4.9308, p = 0.0264) and species distribution (X 2 = 28.3236, P = 0.0001), the results indicated that FMD is highly prevalent in wildlife-livestock interface areas than in non-interface areas. However, uncontrolled livestock movement in Kongwa District resulted into much higher FMD prevalence than in districts where there is wildlife-livestock interface. The presence of antibodies against FMD virus in species other than cattle revealed that there is a need to consider other species in planning for FMD control

Acquisition of antibodies to merozoite surface protein 3 among residents of Korogwe, north eastern Tanzania

<p>Abstract</p> <p>Background</p> <p>A polymorphic malaria parasite antigen, merozoite surface protein 3 (MSP3), is among the blood stage malaria vaccine candidates. It is believed to induce immunity through cytophilic antibodies that disrupt the process of erythrocytes invasion by merozoites. This study aimed at assessing natural acquisition of antibodies to MSP3 in individuals living in an area with different malaria transmission intensity in preparation for malaria vaccine trials.</p> <p>Methods</p> <p>The study was conducted in individuals aged 0-19 years from villages located in lowland, intermediate and highland strata in Korogwe district, northeastern Tanzania. Blood samples from 492 study participants were collected between May and June 2006 for malaria diagnosis and immunological investigations. Reactivity of MSP3 to different types of antibodies (immunoglobulin M, G and IgG subclass 1 and 3) were analysed by Enzyme Linked ImmunoSorbent Assay (ELISA).</p> <p>Results</p> <p>Malaria parasite prevalence was higher in the lowland (50%) compared to the intermediate (23.1%) and highland (9.8%) strata. Immunogloblin G subclasses 1 and 3 (IgG1 & IgG3), total IgG and IgM were found to increase with increasing age. IgG3 levels were significantly higher than IgG1 (p < 0.001). Furthermore, <it>Plasmodium falciparum </it>infection was associated with higher IgG3 levels (p = 0.008). Adjusting by strata and age in individuals who had positive blood smears, both IgG and IgM were associated with parasite density, whereby IgG levels decreased by 0.227 (95%CI: 0.064 - 0.391; p = 0.007) while IgM levels decreased by 0.165 (95%CI: 0.044 - 0.286; p = 0.008).</p> <p>Conclusion</p> <p>Individuals with higher levels of IgG3 might be partially protected from malaria infection. Higher levels of total IgG and IgM in highlands might be due to low exposure to malaria infection, recent infection or presence of cross-reactive antigens. Further studies of longitudinal nature are recommended. Data obtained from this study were used in selection of one village (Kwashemshi) for conducting MSP3 phase 1b malaria vaccine trial in Korogwe.</p

Satisfactory safety and immunogenicity of MSP3 malaria vaccine candidate in Tanzanian children aged 12-24 months.

BACKGROUND: Development and deployment of an effective malaria vaccine would complement existing malaria control measures. A blood stage malaria vaccine candidate, Merozoite Surface Protein-3 (MSP3), produced as a long synthetic peptide, has been shown to be safe in non-immune and semi-immune adults. A phase Ib dose-escalating study was conducted to assess the vaccine's safety and immunogenicity in children aged 12 to 24 months in Korogwe, Tanzania (ClinicalTrials.gov number: NCT00469651). METHODS: This was a double-blind, randomized, controlled, dose escalation phase Ib trial, in which children were given one of two different doses of the MSP3 antigen (15 microg or 30 microg) or a control vaccine (Engerix B). Children were randomly allocated either to the MSP3 candidate malaria vaccine or the control vaccine administered at a schedule of 0, 1, and 2 months. Immunization with lower and higher doses was staggered for safety reasons starting with the lower dose. The primary endpoint was safety and reactogenicity within 28 days post-vaccination. Blood samples were obtained at different time points to measure immunological responses. Results are presented up to 84 days post-vaccination. RESULTS: A total of 45 children were enrolled, 15 in each of the two MSP3 dose groups and 15 in the Engerix B group. There were no important differences in reactogenicity between the two MSP3 groups and Engerix B. Grade 3 adverse events were infrequent; only five were detected throughout the study, all of which were transient and resolved without sequelae. No serious adverse event reported was considered to be related to MSP3 vaccine. Both MSP3 dose regimens elicited strong cytophilic IgG responses (subclasses IgG1 and IgG3), the isotypes involved in the monocyte-dependant mechanism of Plasmodium falciparum parasite-killing. The titers reached are similar to those from African adults having reached a state of premunition. Furthermore, vaccination induced seroconversion in all vaccinees. CONCLUSION: The MSP3 malaria vaccine candidate was safe, well tolerated and immunogenic in children aged 12-24 months living in a malaria endemic community. Given the vaccine's safety and its induction of cytophilic IgG responses, its efficacy against P. falciparum infection and disease needs to be evaluated in Phase 2 studies

Epidemiology of Malaria in an Area Prepared for Clinical Trials in Korogwe, North-eastern Tanzania.

Site preparation is a pre-requesite in conducting malaria vaccines trials. This study was conducted in 12 villages to determine malariometric indices and associated risk factors, during long and short rainy seasons, in an area with varying malaria transmission intensities in Korogwe district, Tanzania. Four villages had passive case detection (PCD) of fever system using village health workers. Four malariometric cross-sectional surveys were conducted between November 2005 and May 2007 among individuals aged 0-19 years, living in lowland urban, lowland rural and highland strata. A total of 10,766 blood samples were collected for malaria parasite diagnosis and anaemia estimation. Blood smears were stained with Giemsa while haemoglobin level was measured by HaemoCue. Socio-economic data were collected between Jan-Apr 2006. Adjusting for the effect of age, the risk of Plasmodium falciparum parasitaemia was significantly lower in both lowland urban, (OR = 0.26; 95%CI: 0.23-0.29, p < 0.001) and highlands, (OR = 0.21; 95%CI: 0.17-0.25, p < 0.001) compared to lowland rural. Individuals aged 6-9 years in the lowland rural and 4-19 years in both lowland urban and highlands had the highest parasite prevalence, whilst children below five years in all strata had the highest parasite density. Prevalence of splenomegaly and gametocyte were also lower in both lowland urban and highlands than in lowland rural. Anaemia (Hb <11 g/dl) prevalence was lowest in the lowland urban. Availability of PCD and higher socio-economic status (SES) were associated with reduced malaria and anaemia prevalence. Higher SES and use of bed nets in the lowland urban could be the important factors for low malaria infections in this stratum. Results obtained here were used together with those from PCD and DSS in selecting a village for Phase 1b MSP3 vaccine trial, which was conducted in the study area in year 2008

Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa

Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent

Designing and development of multiplex rapid diagnostic test platforms for health-related point-of-care applications in the developing world

A Dissertation Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Life Sciences and Bioengineering of the Nelson Mandela African Institution of Science and TechnologyAccurate and timely diagnosis is usually an important step towards appropriate disease management. In resource-limited settings, healthcare workers lack proper facilities to perform vital tests, and the diagnosis of disease is often determined by non-specific, physiological symptoms alone. Simple and rapid tests are needed as pre-requisite tools for patient care and must conform to the criteria set out by the World Health Organization (WHO). Such tests should be affordable, sensitive, specific, user-friendly, robust, equipment-free, and deliverable to the needy, and abbreviated as ASSURED diagnostics. To address the challenge of specific diagnosis for diseases that present with similar symptoms, multiplex diagnostic platforms must be designed. The use of paper has been extensively studied for its potential as matrix for microfluidic devices. In fabrication of multiplexed microfluidic devices, patterning is an important step. Various approaches including cutting, photolithography, wax-printing, plotting and etching have been developed and tested on paper. In recent years, the focus has also been directed towards exploring the potential of hydrophilic threads as a convenient and low-cost approach for fabrication of microfluidic channels and as signal substrate. This research was aimed at designing multiplex diagnostic test platforms that could be used at point-of-care especially in resource-limited settings. The first approach was to design multiplex paper-based platforms where paper was patterned using a combination of cutting and wax-printing. In the second approach, ordinary thread was used to create microfluidic channels and paper discs were used for sample loading, reagent storage and results display to develop a colorimetric test platform that was demonstrated using glucose, uric acid and bovine serum albumin. In the third design, thread was used in combination with nitrocellulose membrane to develop an immunochromatographic test platform for infectious diseases diagnosis and was tested using Helicobacter pylori (bacteria), Hepatitis B surface antigen (viral antigen), and Immunoglobulin G (antibody). Thread-based designs were evaluated for short-term viability under normal storage conditions. All designs produced rapid results with the lowest limit of detection for antigens being as low as 30 ng/ml for immunochromatographic tests. Treatment of hydrophobic threads improved their wicking, with mercerization being the most efficient with wicking rate up to 42 cm/minute. Reagents, including enzymes and antibodies were found to remain viable in dried form on membranes and paper discs. Developing multiplexed paper- and thread-based diagnostic platforms offers a potential approach in developing ASSURED diagnostics to address health challenges in resource-limited settings. Efforts should be directed towards transforming the proposed designs into viable products that can be used at point-of-care

Challenges in Diagnosis of Febrile illnesses in Tanzania in the Era of Declining Malaria Epidemiology

Research Article published by American Journal of Research Communication Vol 3(5)Malaria and other febrile illnesses are very common especially in children in developing countries. Due to reliance on clinical algorithms for diagnosis in resource-poor settings, most febrile episodes have always been attributed to malaria. However, continuous malaria monitoring and recent improvements in malaria diagnosis have revealed a progressive decline in malaria and significant involvement of non-malarial etiologies in most febrile cases. This paper highlights the situation of malarial and non-malarial fevers, challenges facing the health sector, and possible approaches to addressing these challenges for better diagnosis of non-malarial febrile illnesses in Tanzania

Review of Peste des Petits Ruminants Occurrence and Spread in Tanzania

Peste des petits ruminants (PPR) is an important transboundary animal disease of domestic small ruminants, camels, and wild artiodactyls. The disease has significant socio-economic impact on communities that depend on livestock for their livelihood and is a threat to endangered susceptible wild species. The aim of this review was to describe the introduction of PPR to Tanzania and its subsequent spread to different parts of the country. On-line databases were searched for peer-reviewed and grey literature, formal and informal reports were obtained from Tanzanian Zonal Veterinary Investigation Centres and Laboratories, and Veterinary Officers involved with PPR surveillance were contacted. PPR virus (PPRV) was confirmed in northern Tanzania in 2008, although serological data from samples collected in the region in 1998 and 2004, and evidence that the virus was already circulating in Uganda in 2003, suggests that PPRV might have been present earlier than this. It is likely that the virus which became established in Tanzania was introduced from Kenya between 2006–7 through the cross-border movement of small ruminants for trade or grazing resources, and then spread to eastern, central, and southern Tanzania from 2008 to 2010 through movement of small ruminants by pastoralists and traders. There was no evidence of PPRV sero-conversion in wildlife based on sera collected up to 2012, suggesting that they did not play a vectoring or bridging role in the establishment of PPRV in Tanzania. PPRV lineages II, III and IV have been detected, indicating that there have been several virus introductions. PPRV is now considered to be endemic in sheep and goats in Tanzania, but there has been no evidence of PPR clinical disease in wildlife species in Tanzania, although serum samples collected in 2014 from several wild ruminant species were PPRV sero-positive. Similarly, no PPR disease has been observed in cattle and camels. In these atypical hosts, serological evidence indicates exposure to PPRV infection, most likely through spillover from infected sheep and goats. Some of the challenges for PPRV eradication in Tanzania include movements of small ruminants, including transboundary movements, and the capacity of veterinary services for disease surveillance and vaccination. Using wildlife and atypical domestic hosts for PPR surveillance is a useful indicator of endemism and the ongoing circulation of PPRV in livestock, especially during the implementation of vaccination to control or eliminate the disease in sheep and goats. PPR disease has a major socio-economic impact in Tanzania, which justifies the investment in a comprehensive PPRV eradication programme