Biology subjects

Cunningham, L. J.

Publications and source records attributed to Cunningham, L. J..

6 recordsLinked to original sources

bioRxiv2025

Development and pilot application of a point-of-need molecular xenomonitoring protocol for tsetse (Glossina sp.) in a low-resource setting

BackgroundTsetse flies (Glossina sp.) are the primary vectors of trypanosomes causing human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). Disease surveillance can be carried out by detecting Trypanosoma DNA in tsetse, also known as molecular xenomonitoring. Whilst molecular methods can increase the efficiency and sensitivity of pathogen detection, trained staff and a well-equipped laboratory are required. In many cases, DNA extraction and screening is outsourced to a central laboratory in a major city either in-country or abroad, far removed from original tsetse collection sites. This increases results turnaround time, incurs transportation costs, and can lead to sample loss or damage. Methodology/Principle FindingsWe set out to develop, optimise and trial methods for tsetse xenomonitoring in a low-resource point-of-need setting. A low-cost protocol was developed consisting of rapid alkali-based DNA extraction and Trypanosoma detection qPCR assays using air-dryable reagent mixes. A minimally-equipped laboratory was established in a field station in Arua, Uganda. Following a training workshop, three entomology technicians carried out screening on 286 tsetse collected over a nine-week study period. The technicians consistently extracted high quality DNA (98% success rate) and were able to successfully detect T. brucei sensu lato, T. congolense and T. vivax DNA in 3.6% - 4.3% (95% confidence interval [1.73, 7.73]) of total tsetse. Conclusions/SignificanceThis study demonstrated that sensitive molecular xenomonitoring of HAT and AAT pathogens can be carried out without the need for cold-chain storage or high-powered equipment. Further improvements to the system might be achieved by modifying the DNA extraction protocol to enable high-throughput or pooled samples, increasing the sensitivity of the T. b. gambiense DNA detection assay and exploring more sustainable power sources. Author SummaryTsetse flies spread the parasitic diseases human African trypanosomiasis (sleeping sickness) and animal African trypanosomiasis (nagana) that impact populations across sub-Saharan Africa. Disease surveillance can be carried out using tests to detect parasite DNA in tsetse, termed molecular xenomonitoring. Currently, these methods are too complex, costly and logistically-challenging to be carried out in remote areas where sleeping sickness is a problem. However, advances in molecular testing technology are now making this a possibility. We set out to develop a tsetse molecular xenomonitoring system using a basic laboratory set-up in Arua, Uganda. The protocol comprised a low-cost method to extract DNA from tsetse, a portable qPCR machine to test samples and air-dried reagents that did not require cold storage. Following a two-week training workshop, three technicians went on to carry out testing on 286 tsetse over a nine-week period. The technicians were able to consistently extract high-quality DNA (98% success rate) and successfully detected trypanosome parasite DNA in 30 (10.7%) tsetse samples. Whilst there are still challenges to overcome, this study has demonstrated that molecular xenomonitoring of tsetse can be carried out without the need for trainees with previous molecular experience, refrigerated reagents or high-powered equipment.

molecular biology↗

bioRxiv2024

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection

BackgroundTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naive tsetse with T. brucei DNA via faeces when co-housed. Methodology/Principle FindingsInsectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naive tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naive tsetse also tested positive. Even at a ratio of 1 infected to 11 naive flies, 91% of naive tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naive tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/SignificanceOur results show that infected tsetse can contaminate naive flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives. Author SummaryTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei parasites that cause human African trypanosomiasis, also known as sleeping sickness. As part of disease surveillance, tsetse can be captured in traps and checked for parasite presence. The molecular screening of disease vectors (such as mosquitoes, ticks and blackflies) for the presence of pathogen DNA has gained popularity in recent years. However, DNA contamination may occur at capture when live vectors are retained for a limited period in a trap cage. To explore this, we conducted experiments, initially with laboratory-reared tsetse and then field-caught tsetse from Tanzania. Our results show that infected tsetse can contaminate uninfected tsetse with T. brucei DNA when retained together in a trap cage, and that the level of contamination can be extensive. Infected tsetse consistently shed T. brucei DNA in their faeces, which in turn contaminates other tsetse. This can produce false-positive results, leading to inaccurate reporting of infection prevalence. These findings impact not only trypanosomiasis surveillance, but may also have ramifications for the xenomonitoring of other vector-borne neglected diseases. Future work should explore whether pathogen DNA contamination routes exist in other vector species and, if so, the methods to mitigate DNA contamination in entomological traps.

molecular biology↗

bioRxiv2023

Insights into Trypanosomiasis Transmission: Age, Infection Rates and Bloodmeal Analysis of Glossina fuscipes fuscipes in N.W. Uganda

BackgroundTsetse flies (Glossina) transmit species of Trypanosoma which cause human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). Measuring the infection status of wild-caught tsetse is an important part of operations to control HAT. In north-west Uganda, we conducted field studies over a 15-month period to compare classical, microscope-based, and PCR-based methods of detecting trypanosomes in tsetse. We also quantified the age structure and host preferences of tsetse. MethodsUsing Pyramidal traps placed along the Kochi River, in Koboko district, 12512 G. fuscipes fuscipes were caught. A subset of females ({not}n= 5051) and males (n= 1221) underwent dissection wherein mouthparts, midguts and salivary glands were screened for trypanosomes. Additionally, the age of the females was estimated using ovarian ageing and the trypanosome-positive status of 1931 females and 438 males was investigated by ITS PCR. Further the bloodmeal sources of 131 tsetse were identified using vertebrate cytochrome b PCR. ResultsInfection rates estimated were significantly greater (1.9-9.3 times) using the PCR-based method compared to the classical dissection-based method. Positive rates for T. brucei sl, T. congolense and T. vivax were 1.6% (1.32-2.24), 2.4% (1.83-3.11and 2.0% (1.46-2.63), respectively by PCR. The abundance and age structure of tsetse populations were relatively stable and the slight seasonal four-fold variation in abundance appeared to be correlated with rainfall. Analyses of age structure suggests a low natural daily mortality of 1.75% (1.62-1.88). The majority of bloodmeals were identified as cattle (39%, 30.5-47.8) and human (37% of meals, 28.4-45.6). ConclusionPCR provides a more sensitive and specific method of estimating the infection rates of all pathogenic species of trypanosome circulating in Koboko. The seasonally stable abundance, low mortality rate and high proportion of bloodmeals from humans may explain, in part, why this district has historically been a focus of sleeping sickness. Author summaryTsetse flies (Glossina) transmit African trypanosomiasis in humans ( sleeping sickness) and livestock ( nagana). Identifying trypanosome species and quantifying infection rates in tsetse is important in understanding and controlling trypanosomiasis. Traditionally this relied on microscopic examination of tsetse, however, this method is unable to reliably identify different species of trypanosome. We compared microscopy and PCR, trypanosome-detection methods, using G. fuscipes fuscipes collected from traps deployed in Koboko district, in North-West Uganda. Our results show that the PCR-based method was 1.9-9.3 times more sensitive than the classical approach and able to identify different species of Trypanosoma, including mixed infections. In collecting tsetse we also obtained data on the seasonal abundance, age and diet of the tsetse population. Analyses of these results showed that natural mortality was low (1.75%/day) among adult females and only a slight seasonal variation in abundance and age structure occurred. The most important hosts were cattle and humans, providing 39% and 37% of bloodmeals, respectively. The PCR method provided a sensitive means of identifying trypanosomes to the species level. The longevity and diet of tsetse in Koboko may explain, why this district was a persistent focus of disease prior to the deployment of Tiny Targets to control tsetse.

ecology↗

bioRxiv2020

Impact of vector control on effective population sizes; empirical evidence for a control- based genetic bottleneck in the tsetse fly Glossina fuscipes

We investigated genetic variation at 37 newly-developed microsatellite loci in populations of the tsetse fly Glossina fuscipes fuscipes captured from the upper and lower reaches of a single hydrographical network within an endemic Human African Trypanosomiasis focus. Our primary aim was to assess the impact of vector control using insecticide-treated baits (Tiny Targets) on genetic structure. We initially used STRUCTURE to delineate geographical boundaries of two stable ancestral reference populations without any history of vector control but marked for either vector control ( intervention) or no control ( non-intervention). We then used the ADMIXTURE model to assess genetic divergence in temporal populations collected after vector control implementation. We applied the Linkage Disequilibrium method to explicitly measure spatial and temporal changes in effective population size (Ne). We observed a significant reduction in Ne coincident with vector control, whereas Ne remained stable in the non-intervention area. Our empirical findings show how classical population genetics approaches detected within a short period of time, a significant genetic bottleneck associated with vector control, and opens up the possibility of using routine genomic surveillance. We have also generated a resource of new genetic markers for studies on the population genetics of tsetse at finer-scale resolution. FundingThis work was funded through a Wellcome Trust Masters Fellowship in Public Health and Tropical Medicine awarded to Allan Muhwezi (103268/Z/13/Z).

evolutionary biology↗

bioRxiv2020

Identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using multiplexed high-resolution melt analysis.

BackgroundHuman African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term Rhodesian HAT. Here we describe the development of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. MethodsPrimers for T. b. rhodesiense and T. b. gambiense were designed to target species-specific single copy genes. An additional primer set was included in the multiplex to determine if samples have sufficient genomic material for detecting low copy number targets. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were infected with T. b. rhodesiense. ResultsThe assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. HRM successfully identified three T. b. rhodesiense positive flies and was in agreement with the reference sub-species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l and produces results in ~2 hours, avoiding gel electrophoresis. ConclusionsThis method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

molecular biology↗

bioRxiv2019

Evidence of the absence of Human African Trypanosomiasis in northern Uganda: analyses of cattle, pigs and tsetse flies for the presence of Trypanosoma brucei gambiense

BackgroundLarge-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda.\n\nMethodsWe collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples.\n\nResultsIn total, 39/2,088 (1.9%; 95% CI=1.9-2.5) cattle, 25/400 (6.3%; 95% CI=4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI=1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense.\n\nConclusionThe absence of T. b. gambiense in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.\n\nAuthor SummaryThe decline of annual cases of West-African sleeping sickness in Uganda raises the prospect that elimination of the disease is achievable for the country. However, with the decrease in incidence and the likely subsequent change in priorities there is a need to confirm that the disease is truly eliminated. One unanswered question is the role that domestic animals play in maintaining transmission of the disease. The potential of cryptic-animal reservoirs is a serious threat to successful and sustained elimination of the disease. It is with the intent of resolving this question that we have carried out this study whereby we examined 2088 cattle, 400 pigs and 2184 tsetse for Trypanosoma brucei gambiense, the parasite responsible for the disease. Our study found T. brucei s.l. in local cattle, pigs and tsetse flies, with their respective prevalences as follows, 1.9%, 6.3% and 1.8%. Further analysis to establish identity of these positives to the sub-species level found that no cattle, pigs or tsetse were carrying the pathogen responsible for Gambian sleeping sickness. Our work highlights the difficulty of establishing the absence of a disease, especially in an extremely low endemic setting, and the limitations of some of the most commonly used methods.

microbiology↗