Card Agglutination Test for Trypanosomiasis (CATT) (7)

• Variant antigen LiTat 1.3• Sensitivity: 87-98%

Latex Agglutination test (LATEX) (7)

• Variant antigens LiTat 1.3, 1.5, 1.6• Sensitivity: 71-100%

Quantitative Buffy Coat (QBC) test (8)

• Detection of trypomastigostes• Sensitivity: 95% for 450

parasites/ml blood

Microscopic observation – blood samples (8)

• Thin blood film – Detection limit: 6000-10,000 parasites/ml

• Thick blood film – Detection limit: 600-5,000 parasites/ml

Second Stage Diagnostics – Analysis of Cerebrospinal Fluid (8,9)

• White blood cell count:Early Second Stage: 6-20 cells/mlLate Second Stage: 11-20 cells/ml and presence of antibodies (IgM)• Trypanosoma detection.

CURRENT DIAGNOSTICS AND LIMITATIONS

Figure 7. Detection of trypanosoma by observation of the agglutination in 7 samples with different concentrations of the parasite. (12)

Figure 8. Trypamastigotes can be detected in the buffy coat of centrifuged blood. (13)

Figure 9. Thin blood film. (14)

Figure 10. Thick blood film. (14)

Figure 11 : Cartoon diagram of T.brucei species in black. Available at : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278896/figure/F1/

T.brucei flagellum morphology

Combating African Sleeping Sickness by Developing Novel Diagnostic Strategies

By Andreas Hadjicharalambous, Carina Conceição and Meghan Place

This device makes possible an early diagnosis for African sleeping sickness and therefore provides a higher probability for the survival of infected individuals:

• Sufficiently low cost of manufacturing

• Accurate results within 20 minutes

• Could be used in many areas of the developing world

• Requires no medical skills to be applied

Parasite on a chip

This device puts the lab on a chip and it is based on a microfluidic system. These systems consist of micro channels in which a certain volume of fluid travels from point A to point D and is analyzed through the components in that mixture.

The Device Compartments

• A plastic chip with micro channels approximately 12 cm in length

• Plastic tubes for loading the blood sample and chemical reagents

• A plastic syringe

How does it work ?

This device focuses on the detection of a particular parasite, giving an accurate result within 20 minutes . The stages of each detection test are explained below:

A. Approximately 1ml of blood is transferred to the indicated area in order for the test to be initiated

B. The blood sample passes through a nanostructure filter which size-excludes undesirable molecules

C. The blood sample components (possibly including parasites) react with the genetically engineered molecule HpHbTLF-1, which is attached to nanobodies specific for the paraflagellar rod protein of Trypanosoma brucei (16)

D. Parasites enter a circular maze where they attach to the wall structure by binding into specific RNA aptamers which will be specific for the enzymes present on the flagellum.

Detection System

• Once parasites are captured on the wall of the micro channels, a washing reagent of secondary nanobodies attached with gold nanoparticles bind to the “trapped” parasites.

• A silver development reagent is then applied, which reacts with the captured nanoparticles and coats them with silver. After approximately 20 minutes , a thick silver film is formed in the circle reflecting the amount of nanobodies attached and therefore the presence and adundance of the parasite.

• Labeling HpHbTLF-1 following a luciferase reporter assay, Trypanosome spp. could not only be observed but also be differentiated as T.b.gambiense or T.b.rhodesiense

INTRODUCING THE NEW DIAGNOSTIC DEVICE

DISCUSSION

Trypanosomiasis

Caused by several species of Trypanosoma2

T. brucei rhodesiense T. brucei gambiense

Vector: Glossina genus

Figure 1. A false-colored scanning electron microscope image of an African trypanosome.3

Figure 2. Glossina spp.4

Figure 3. Distribution of Human African Figure 4. Blood smear of T. brucei.6

trypanosomiasis (T.b. gambiense) in 2013.5

Variant Surface Glycoprotein Coat

Figure 5. Antigenic variation in T .brucei Figure 6. Computer image of a

is used to evade the immune system.7 variant surface glycoprotein.8

2 stages of disease1

1. haemolymphatic phase 2. neurological or

meningoencephalic phase

Results in headache, sleep disturbances, drowsiness, abnormal neurological function, coma, and death.

Without treatment, African Sleeping Sickness is considered 100% fatal

Current treatment: First stage: Pentamidine, Suramin Second stage: Melarsoprol, Eflornithine,

Nifurtimox and Eflornithine combination

Early diagnosis and treatment is essential

INTRODUCTION

A

B

C D

Detection Area

Reaction Area

Figure 12 : Diagram showing the role of TbHpHbR, Hp and Hb as well as the mechanism of action of Apol! And SRA . Available at : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278896/figure/F1/

= TbHpHbTLF-1

= RNA Aptamer

1. World Health Organization (2013) “Trypanosomiasis, human African (sleeping sickness)” Available at: http://www.who.int/mediacentre/factsheets /fs259/en/.

2. Bauman, Robert W. (2014) Microbiology with Diseases by Taxonomy (p. 663-664). 4th edition. Glenview, IL: Pearson Education.

3. American Society for Microbiology (2011) “Eukaryotic Cell: cover image” Available at: http://ec.asm.org/content/10/7.cover-expansion.

4. The Guardian (2011) “Putting sleeping sickness on the radar” Available at: http://www.theguardian.com/education/2011/sep/19/max-perutz-science-award-winner.

5. World Health Organization (2013) “Global Health Observatory Map Gallery” Available at: http://gamapserver.who.int/mapLibrary/app/searchResults.aspx.

6. Microbe Wiki (2013) “Membrane proteins as mechanisms of immune system evasion in Trypanosoma brucei” Available at: https://microbewiki.kenyon.edu/index.php/Membrane_proteins_as_mechanisms _of_immune_system_evasion_in_Trypanosoma_brucei.

7. Rudenko Lab “Research introduction: African Trypanosomes” Available at http://rudenkolab.co.uk/Research-intro.html.

8. Barry, Dave and Carrington, Mark. University of Glasgow: WCMP “Antigenic Variation and the Variant Surface Glycoprotein (VSG)” Available at: http://leishman.cent.gla.ac.uk/kook_about.html.

9. Truc P, Lejon V, Magnus E, Jamonneau V, Nangouma A, Verloo D, Penchenier L and Büscher P. 2002. Evaluation of the micro-CATT, CATT/Trypanosoma brucei gambiens, and LATEX/T. b. gambiense methods for serodiagnosis and surveillance of human African trypanosomiasis in West and Central Africa. Bulletin of the World Health Organization, 80 (11).

10. Chappuis F, Loutan L, Simarro P, Lejon V and Büscher P. 2005. Options for Field Diagnosis of Human African Trypanosomiasis. Clinical Microbiology Reviews: Vol. 18, p. 133-146.

11. Diagnostics test for African trypanosomiasis, available at: http://bestpractice.bmj.com/best-practice/monograph/9999/diagnosis/tests.html.

12. CATT test, available at: http://wellcomeimages.org/indexplus/result.html?*sform=wellcome-images&_IXACTION_=query&%24%3 Dtoday=&_IXFIRST_=1&%3Did_ref=W0050869&_IXSPFX_=templates/t&_IXFPFX_=templates/t&_IXMAXHITS_=1.

13. QBC test, available at: https://www.geisingermedicallabs.com/catalog/blood_specimens.shtml.

14. Blood Films, available at: http://pt.slideshare.net/tpgmedical/preparation-of-blood-films-for-malaria-parasites.

15. Obishakin E, Stijlemans B, Santi-Rocca J, et al. Generation of a Nanobody Targeting the Paraflagellar Rod Protein of Trypanosomes. Tetteh KKA, ed. PLoS ONE 2014;9(12):e115893. doi:10.1371/journal.pone.0115893. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles /PMC4281110/.

16. Langousis, G., & Hill, K. L. (2014). Motility and more: the flagellum ofTrypanosoma brucei. Nature Reviews. Microbiology, 12(7), 505–518. doi:10.1038/nrmicro3274. Available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278896/.

17. Richard J.Wheeler (2014). The trypanolytic factor-mechanism, impacts and applications.Trends in Parasitology, Volume 26, Issue 9, September 2010, Pages 457–464. Available at: http://www.sciencedirect.com/science/article/pii/S1471492210001078#gr3.

REFERENCES