scholarly journals A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR

2020 ◽  
Vol 14 (11) ◽  
pp. e0008308
Author(s):  
Gala Garrod ◽  
Emily R. Adams ◽  
Jessica K. Lingley ◽  
Isabel Saldanha ◽  
Stephen J. Torr ◽  
...  
Keyword(s):  
High Resolution ◽  
Trypanosoma Brucei ◽  
Limit Of Detection ◽  
Parasitic Infection ◽  
Screening Tools ◽  
Tsetse Flies ◽  
High Resolution Melt ◽  
Subjective Analysis ◽  
Trypanosoma Brucei Gambiense ◽  
Species Specific

Human 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 for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The 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. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference 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 less than 2 hours, avoiding gel electrophoresis and subjective analysis. This 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.

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

2020 ◽  
Author(s):  
Gala Garrod ◽  
Emily R. Adams ◽  
Jessica K. Lingley ◽  
Isabel Saldanha ◽  
Stephen J. Torr ◽  
...  
Keyword(s):  
High Resolution ◽  
Trypanosoma Brucei ◽  
Limit Of Detection ◽  
Parasitic Infection ◽  
Cross Reactivity ◽  
Screening Tools ◽  
Tsetse Flies ◽  
High Resolution Melt ◽  
Trypanosoma Brucei Gambiense ◽  
Species Specific

AbstractBackgroundHuman 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.

Escape mechanisms of African trypanosomes: why trypanosomosis is keeping us awake

Parasitology ◽  
2014 ◽  
Vol 142 (3) ◽  
pp. 417-427 ◽  
Author(s):  
JENNIFER CNOPS ◽  
STEFAN MAGEZ ◽  
CARL De TREZ
Keyword(s):  
Immune Response ◽  
Trypanosoma Brucei ◽  
Parasitic Infection ◽  
Mammalian Host ◽  
Tsetse Flies ◽  
African Trypanosomes ◽  
Transmission Potential ◽  
Highly Sensitive ◽  
Commercially Important ◽  
And Control

SUMMARYAfrican trypanosomes have been around for more than 100 million years, and have adapted to survival in a very wide host range. While various indigenous African mammalian host species display a tolerant phenotype towards this parasitic infection, and hence serve as perpetual reservoirs, many commercially important livestock species are highly disease susceptible. When considering humans, they too display a highly sensitive disease progression phenotype for infections withTrypanosoma brucei rhodesienseorTrypanosoma brucei gambiense, while being intrinsically resistant to infections with other trypanosome species. As extracellular trypanosomes proliferate and live freely in the bloodstream and lymphatics, they are constantly exposed to the immune system. Due to co-evolution, this environment however no longer poses a hostile threat, but has become the niche environment where trypanosomes thrive and obligatory await transmission through the bites of tsetse flies or other haematophagic vectors, ideally without causing severe side infection-associated pathology to their host. Hence, African trypanosomes have acquired various mechanisms to manipulate and control the host immune response, evading effective elimination. Despite the extensive research into trypanosomosis over the past 40 years, many aspects of the anti-parasite immune response remain to be solved and no vaccine is currently available. Here we review the recent work on the different escape mechanisms employed by African Trypanosomes to ensure infection chronicity and transmission potential.

scholarly journals Screening of Trypanosoma brucei gambiense in Domestic Livestock and Tsetse Flies from an Insular Endemic Focus (Luba, Equatorial Guinea)

2010 ◽  
Vol 4 (6) ◽  
pp. e704 ◽  
Author(s):  
Carlos Cordon-Obras ◽  
Carmen García-Estébanez ◽  
Nicolás Ndong-Mabale ◽  
Simón Abaga ◽  
Pedro Ndongo-Asumu ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Equatorial Guinea ◽  
Tsetse Flies ◽  
Trypanosoma Brucei Gambiense ◽  
Endemic Focus ◽  
Domestic Livestock ◽  
Insular Endemic

scholarly journals Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

2020 ◽  
Vol 48 (7) ◽  
pp. e42-e42 ◽  
Author(s):  
Justin C Rolando ◽  
Erik Jue ◽  
Jacob T Barlow ◽  
Rustem F Ismagilov
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Rapid Diagnostics ◽  
High Resolution Melt ◽  
Specific Amplification ◽  
The Impact

Abstract Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

scholarly journals 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

2019 ◽  
Author(s):  
Lucas J. Cunningham ◽  
Jessica K. Lingley ◽  
Iñaki Tirados ◽  
Johan Esterhuizen ◽  
Mercy A. Opiyo ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Large Scale ◽  
Human African Trypanosomiasis ◽  
Domestic Animals ◽  
Sleeping Sickness ◽  
Tsetse Flies ◽  
Microscopical Examination ◽  
African Trypanosomiasis ◽  
Trypanosoma Brucei Gambiense ◽  
Reservoir Hosts

AbstractBackgroundLarge-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.MethodsWe 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.ResultsIn 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.ConclusionThe 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.Author 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.

scholarly journals Trypanosomal variant surface glycoprotein expression in human African trypanosomiasis patients

2021 ◽  
Author(s):  
Jaime So ◽  
Sarah Sudlow ◽  
Abeer Sayeed ◽  
Tanner Grudda ◽  
Stijn Deborggraeve ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Tsetse Flies ◽  
African Trypanosomiasis ◽  
Trypanosoma Brucei Gambiense ◽  
Terminal Domain ◽  
Human Infections

AbstractTrypanosoma brucei gambiense, an extracellular protozoan parasite, is the primary causative agent of human African Trypanosomiasis. T. b. gambiense is endemic to West and Central Africa where it is transmitted by the bite of infected tsetse flies. In the bloodstream of an infected host, the parasite evades antibody recognition by altering the Variant Surface Glycoprotein (VSG) that forms a dense coat on its cell surface through a process known as antigenic variation. Each VSG has a variable N-terminal domain that is exposed to the host and a less variable C-terminal domain that is at least partially hidden from host antibodies. Our lab developed VSG-seq, a targeted RNA-seq method, to study VSG expression in T. brucei. Studies using VSG-seq to characterize antigenic variation in a mouse model have revealed marked diversity in VSG expression within parasite populations, but this finding has not yet been validated in a natural human infection. Here, we used VSG-seq to analyze VSGs expressed in the blood of twelve patients infected with T. b. gambiense. The number of VSGs identified per patient ranged from one to fourteen and, notably, two VSGs were shared by more than one patient. Analysis of expressed VSG N-terminal domain types revealed that 82% of expressed VSGs encoded a type B N-terminus, a bias not seen in datasets from other T. brucei subspecies. C-terminal types in T. b. gambiense infection were also restricted. These results demonstrate a bias either in the underlying VSG repertoire of T. b. gambiense or in the selection of VSGs from the repertoire during infection. This work demonstrates the feasibility of using VSG-seq to study antigenic variation in human infections and highlights the importance of understanding VSG repertoires in the field.Author SummaryHuman African Trypanosomiasis is a neglected tropical disease largely caused by the extracellular parasite known as Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their dense surface coat of Variant Surface Glycoprotein (VSG). Despite the important role of VSGs in prolonging infection, VSG expression during natural human infections is poorly understood. A better understanding of natural VSG expression dynamics can clarify the mechanisms which T. brucei uses to alter its VSG coat and improve how trypanosomiasis is diagnosed in humans. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that a diverse range of VSGs are expressed in both natural and experimental infections.

Latent Trypanosoma brucei gambiense foci in Uganda: a silent epidemic in children and adults?

Parasitology ◽  
2011 ◽  
Vol 138 (12) ◽  
pp. 1480-1487 ◽  
Author(s):  
S. L. WASTLING ◽  
K. PICOZZI ◽  
C. WAMBOGA ◽  
B. VON WISSMANN ◽  
C. AMONGI-ACCUP ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Agglutination Test ◽  
Time Points ◽  
Trypanosoma Brucei Gambiense ◽  
Specific Pcr ◽  
Latent Stage ◽  
Centrifugation Technique ◽  
Species Specific ◽  
Asymptomatic Phase ◽  
Western Uganda

SUMMARYTrypanosoma brucei gambiense sleeping sickness follows a long asymptomatic phase and persists in ancient foci from which epidemic clinical disease arises. A putative focus of T. b. gambiense infections has been identified, initially in mothers and young children, on the Lake Albert shoreline of Western Uganda leading to mass screening of 6207 individuals in September 2008. T. b. gambiense infections were identified by Card Agglutination Test for Trypanosomiasis (CATT) and sub-species-specific PCR although parasitological methods failed to confirm any patent trypanosome infections. In April 2009, CATT positives were re-visited; diagnosis of individuals by CATT and PCR was unstable over the two time points and parasites remained undetected, even using mini Anion Exchange Centrifugation Technique (mAECT). These observations suggest the possibility of a silent focus of disease, where all infected individuals are in a latent stage, and highlight our limited understanding of the local natural history and disease progression of T. b. gambiense in children and adults.

scholarly journals Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

Parasitology ◽  
2018 ◽  
Vol 145 (13) ◽  
pp. 1733-1738 ◽  
Author(s):  
Lucas J. Cunningham ◽  
J. Russell Stothard ◽  
Mike Osei-Atweneboana ◽  
Samuel Armoo ◽  
Jaco J. Verweij ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Curve Analysis ◽  
Pcr Analysis ◽  
High Resolution Melt ◽  
Middle Income ◽  
Ancylostoma Duodenale ◽  
Species Specific

AbstractWith the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

Sign in / Sign up

Export Citation Format

Share Document