The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system

Background: Diagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR. Results: A comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%). Conclusions: Based on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach

Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene.

BACKGROUND: The expansion of sleeping sickness caused by Trypanosoma brucei rhodesiense beyond its traditional focus in southeast Uganda has been linked with large-scale livestock restocking. To assess the risk presented to the human population by domestic livestock, human-infective T b rhodesiense must be distinguished from non-human-infective T brucei brucei, since both parasites can be present in cattle. We investigated the use of a simple genetic marker to characterise parasites collected from cattle in villages within the new sleeping sickness focus in Soroti District, Uganda. METHODS: 70 T brucei sl samples of known human infectivity status collected from human beings and cattle in Tororo District, Uganda, from 1989 to 1991 were screened for the presence of the human-serum-resistance-associated (SRA) gene by conventional PCR. In 2000-01, blood samples from 200 randomly selected cattle in six villages and two markets in Soroti District were screened for T brucei sl parasites by PCR; positive samples were screened for the presence of the SRA gene. FINDINGS: The SRA gene was present in all 29 samples from patients with sleeping sickness in Tororo District. Of the 41 samples collected from cattle at the same time, the SRA gene was present in the eight samples that tested resistant to human serum in vitro, whereas it was absent from all 33 isolates that were sensitive to human serum in vitro. Of the 200 cattle sampled in Soroti District, we estimated that up to 18% (95% CI 12-23) were infected with T b rhodesiense. INTERPRETATION: Detection of the SRA gene could provide the basis for a simple diagnostic test to enable targeted control of T b rhodesiense in the domestic livestock reservoir, thereby reducing the public-health burden of sleeping sickness in east Africa

Variant Surface Glycoprotein gene repertoires in Trypanosoma brucei have diverged to become strain-specific

Background: In a mammalian host, the cell surface of African trypanosomes is protected by a monolayer of a single variant surface glycoprotein (VSG). The VSG is central to antigenic variation; one VSG gene is expressed at any one time and there is a low frequency stochastic switch to expression of a different VSG gene. The genome of Trypanosoma brucei contains a repertoire of > 1000 VSG sequences. The degree of conservation of the genomic VSG repertoire in different strains has not been investigated in detail.|Results: Eighteen expressed VSGs from Ugandan isolates were compared with homologues (> 40 % sequence identity) in the two available T. brucei genome sequences. Fourteen homologues were present in the genome of Trypanosoma brucei brucei TREU927 from Kenya and fourteen in the genome of T. b. gambiense Dal972 from Cote d'Ivoire. The Ugandan VSGs averaged 71% and 73 % identity to homologues in T. b. brucei and T. b. gambiense respectively. The sequence divergence between homologous VSGs from the three different strains was not random but was more prevalent in the parts of the VSG believed to interact with the host immune system on the living trypanosome.|Conclusion: It is probable that the VSG repertoires in the different isolates contain many common VSG genes. The location of divergence between VSGs is consistent with selection for strain-specific VSG repertoires, possibly to allow superinfection of an animal by a second strain. A consequence of strain-specific VSG repertoires is that any vaccine based on large numbers of VSGs from a single strain will only provide partial protection against other strains

The diagnosis of trypanosome infections: applications of novel technology for reducing disease risk

Reliable DNA based methodologies to determine prevalence of trypanosome species in domestic livestock have been available for over 10 years. Despite this, they are rarely used to generate baseline data for control operations for these diseases in the field. Rather, such operations tend to rely on data which can be generated using low technology methods such as direct observation of parasites by light microscopy. Here we show the pitfalls of relying on such low tech methodology which, although simple in its application, can provide inaccurate and inadequate data on which to base control methodologies. Our analysis of 61 cattle selected for trypanosome carrier status by either microscopy, low PCV or poor condition score, showed that 90% were infected with trypanosomes while 84% of the total were infected with T. brucei. Diagnosis by PCR on buffy coat preparations on Whatman® FTA® matrices was the most sensitive methodology relative to the gold standard, whereas microscopy was the least sensitive. (African Journal of Biotechnology: 2002 1(2): 39-45

Amplified fragment length polymorphism (AFLP) analysis of closely related wild and captive tsetse fly (Glossina morsitans morsitans) populations

<p>Abstract</p> <p>Background</p> <p>Tsetse flies (Diptera: Glossinidae) are vectors of trypanosomes that cause sleeping sickness in humans and nagana in livestock across sub-Saharan Africa. Tsetse control strategies rely on a detailed understanding of the epidemiology and ecology of tsetse together with genetic variation within and among populations. High-resolution nuclear genetic markers are useful tools for elucidation of the genetic basis of phenotypic traits. In this study amplified fragment length polymorphism (AFLP) markers were developed to analyze genetic variation in <it>Glossina morsitans morsitans </it>from laboratory and field-collected populations from Zimbabwe.</p> <p>Results</p> <p>A total of seven hundred and fifty one loci from laboratory and field populations of <it>G. m. morsitans </it>from Zimbabwe were genotyped using AFLP with seven primer combinations. Analysis identified 335 polymorphic loci. The two populations could be distinguished by cluster and principal components analysis (PCA) analysis, indicating that AFLP markers can be used to separate genetically similar populations; at the same time differences observed between laboratory and field populations were not very great. Among the techniques investigated, the use of acetone was the most reliable method of preservation of tsetse for subsequent extraction of high molecular weight DNA. An interesting finding was that AFLP also enabled robust within-population discrimination of male and female tsetse flies due to their different X chromosome DNA complements.</p> <p>Conclusions</p> <p>AFLP represents a useful additional tool to add to the suite of techniques currently available for the genetic analysis of tsetse populations and represents a useful resource for identification of the genetic basis of important phenotypic traits.</p

Using molecular data for epidemiological inference: assessing the prevalence of Trypanosoma brucei rhodesiense in Tsetse in Serengeti, Tanzania

Background: Measuring the prevalence of transmissible Trypanosoma brucei rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessing human disease risk and monitoring spatio-temporal trends and the impact of control interventions. Although an important epidemiological variable, identifying flies which carry transmissible infections is difficult, with challenges including low prevalence, presence of other trypanosome species in the same fly, and concurrent detection of immature non-transmissible infections. Diagnostic tests to measure the prevalence of T. b. rhodesiense in tsetse are applied and interpreted inconsistently, and discrepancies between studies suggest this value is not consistently estimated even to within an order of magnitude. Methodology/Principal Findings: Three approaches were used to estimate the prevalence of transmissible Trypanosoma brucei s.l. and T. b. rhodesiense in Glossina swynnertoni and G. pallidipes in Serengeti National Park, Tanzania: (i) dissection/microscopy; (ii) PCR on infected tsetse midguts; and (iii) inference from a mathematical model. Using dissection/microscopy the prevalence of transmissible T. brucei s.l. was 0% (95% CI 0–0.085) for G. swynnertoni and 0% (0–0.18) G. pallidipes; using PCR the prevalence of transmissible T. b. rhodesiense was 0.010% (0–0.054) and 0.0089% (0–0.059) respectively, and by model inference 0.0064% and 0.00085% respectively. Conclusions/Significance: The zero prevalence result by dissection/microscopy (likely really greater than zero given the results of other approaches) is not unusual by this technique, often ascribed to poor sensitivity. The application of additional techniques confirmed the very low prevalence of T. brucei suggesting the zero prevalence result was attributable to insufficient sample size (despite examination of 6000 tsetse). Given the prohibitively high sample sizes required to obtain meaningful results by dissection/microscopy, PCR-based approaches offer the current best option for assessing trypanosome prevalence in tsetse but inconsistencies in relating PCR results to transmissibility highlight the need for a consensus approach to generate meaningful and comparable data

Incorporating scale dependence in disease burden estimates:the case of human African trypanosomiasis in Uganda

The WHO has established the disability-adjusted life year (DALY) as a metric for measuring the burden of human disease and injury globally. However, most DALY estimates have been calculated as national totals. We mapped spatial variation in the burden of human African trypanosomiasis (HAT) in Uganda for the years 2000-2009. This represents the first geographically delimited estimation of HAT disease burden at the sub-country scale.Disability-adjusted life-year (DALY) totals for HAT were estimated based on modelled age and mortality distributions, mapped using Geographic Information Systems (GIS) software, and summarised by parish and district. While the national total burden of HAT is low relative to other conditions, high-impact districts in Uganda had DALY rates comparable to the national burden rates for major infectious diseases. The calculated average national DALY rate for 2000-2009 was 486.3 DALYs/100 000 persons/year, whereas three districts afflicted by rhodesiense HAT in southeastern Uganda had burden rates above 5000 DALYs/100 000 persons/year, comparable to national GBD 2004 average burden rates for malaria and HIV/AIDS.These results provide updated and improved estimates of HAT burden across Uganda, taking into account sensitivity to under-reporting. Our results highlight the critical importance of spatial scale in disease burden analyses. National aggregations of disease burden have resulted in an implied bias against highly focal diseases for which geographically targeted interventions may be feasible and cost-effective. This has significant implications for the use of DALY estimates to prioritize disease interventions and inform cost-benefit analyses

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut

A Multi-Host Agent-Based Model for a Zoonotic, Vector-Borne Disease. A Case Study on Trypanosomiasis in Eastern Province, Zambia

Background: This paper presents a new agent-based model (ABM) for investigating T. b. rhodesiense human African trypanosomiasis (rHAT) disease dynamics, produced to aid a greater understanding of disease transmission, and essential for development of appropriate mitigation strategies. Methods: The ABM was developed to model rHAT incidence at a fine spatial scale along a 75 km transect in the Luangwa Valley, Zambia. The method offers a complementary approach to traditional compartmentalised modelling techniques, permitting incorporation of fine scale demographic data such as ethnicity, age and gender into the simulation. Results: Through identification of possible spatial, demographic and behavioural characteristics which may have differing implications for rHAT risk in the region, the ABM produced output that could not be readily generated by other techniques. On average there were 1.99 (S.E. 0.245) human infections and 1.83 (S.E. 0.183) cattle infections per 6 month period. The model output identified that the approximate incidence rate (per 1000 person-years) was lower amongst cattle owning households (0.079, S.E. 0.017), than those without cattle (0.134, S.E. 0.017). Immigrant tribes (e.g. Bemba I.R. = 0.353, S.E.0.155) and school-age children (e.g. 5–10 year old I.R. = 0.239, S.E. 0.041) were the most at-risk for acquiring infection. These findings have the potential to aid the targeting of future mitigation strategies. Conclusion: ABMs provide an alternative way of thinking about HAT and NTDs more generally, offering a solution to the investigation of local-scale questions, and which generate results that can be easily disseminated to those affected. The ABM can be used as a tool for scenario testing at an appropriate spatial scale to allow the design of logistically feasible mitigation strategies suggested by model output. This is of particular importance where resources are limited and management strategies are often pushed to the local scale. © 2016 Alderton et al