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Keywords:

  • buffy coat;
  • diagnosis;
  • Human African trypanosomiasis;
  • mini-anion exchange centrifugation technique (mAECT)
  • trypanosomiase humaine africaine;
  • diagnostic;
  • buffy coat;
  • mAECT
  • Tripanosomiasis humana africana;
  • diagnóstico;
  • capa leucocitaria;
  • mAECT

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Objectives  To evaluate a modification of the mini anion exchange centrifugation test (mAECT) for the diagnosis of Trypanosoma brucei (T.b.) gambiense human African trypanosomiasis (HAT). To increase its sensitivity, this test uses 350 μl of buffy coat withdrawn from 5 ml of blood instead of blood.

Methods  The new protocol was first tested experimentally on serial dilution of trypanosomes and was then further evaluated under field conditions on 57 patients with HAT diagnosed during a medical survey in Guinea.

Results  Experimentally, the use of buffy coats improved mAECT sensitivity at least five fold and enabled to consistently detect parasites in blood at a concentration of 10 trypanosomes/ml. During the field evaluation, more patients tested positive by mAECT-bc (96.5%) than by mAECT-blood (78.9%, χ2 = 6.93, P = 0.008) and lymph juice examination (77.2%, χ2 = 7.67, P = 0.005). Furthermore, the number of parasites per collectors was significantly higher (7.2 vs. 2.6, P = 0.001) when buffy coats were used instead of blood.

Conclusion  The use of the mAECT-bc protocol enabled a significant improvement of HAT parasitological diagnosis in Guinea, without any additional costs. It would deserve to be tested in other T.b. gambiense endemic areas.

Diagnostic de maladie du sommeil: l’utilisation de ‘Buffy Coats’ améliore la sensibilité du test de la mini centrifugation àéchangeuse d’anion

Objectifs:  Evaluer une modification du test de la mini centrifugation àéchangeuse d’anion (mAECT) pour le diagnostic de la trypanosomiase humaine africaine (THA) àTrypanosoma brucei gambiense. Afin d’augmenter sa sensibilité, ce test utilise 350 μl de couenne leuco-plaquettaire (buffy-coat), prélevée sur 5 ml de sang au lieu de sang total.

Méthodes:  Le nouveau protocole a été testé sur des dilutions en série de trypanosomes et a ensuite étéévalué dans des conditions de terrain sur 57 patients THA diagnostiqués lors d’une enquête médicale en Guinée.

Résultats:  Expérimentalement, l’utilisation de ‘buffy coats’ a amélioré la sensibilité du test mAECT d’au moins cinq fois et a permis de détecter de façon consistante des parasites dans le sang à une concentration de 10 trypanosomes/ml. Au cours de l’évaluation sur le terrain, plus de patients ont été testés positifs par mAECT-bc (96,5%) que par mAECT-sang (78,9%; Chi2 = 6,93; p = 0,008) et l’examen du liquide lymphatique (77,2%; Chi2 = 7,67; p = 0,005). Le nombre de parasites par collecteur était significativement plus élevé (7,2 vs 2,6; p = 0,001) lorsque le ‘buffy coats’ plutôt que du sang étaient utilisé.

Conclusion:  L’utilisation du protocole mAECT-bc a permis une amélioration significative du diagnostic parasitologique de la THA en Guinée sans frais supplémentaires. Il mérite d’être testé sur Tb gambiense dans d’autres régions endémiques.

Diagnóstico de la enfermedad del sueño: el uso de la Capa Leucocitaria mejora la sensibilidad de la técnica de filtración en mini-columna de intercambio aniónico (mAECT)

Objetivos:  Evaluar una modificación de la técnica de filtración en mini-columna de intercambio aniónico (mAECT) para el diagnóstico de la tripanosomiasis humana Africana (THA) por Trypanosoma brucei gambiense. Con el fin de aumentar su sensibilidad, se utilizaron 350 μl de la capa leucocitaria (mAECT-cl) tomada a partir de 5 ml de sangre, en vez de sangre (mAECT-s).

Métodos:  El nuevo protocolo se probó primero experimentalmente en una dilución seriada de tripanosomas y después fué evaluado bajo condiciones de campo en 57 pacientes con THA, diagnosticados durante un estudio médico en Guinea.

Resultados:  Experimentalmente, el uso de la capa leucocitaria mejoró la sensibilidad del mAECT en al menos cinco veces, y permitió detectar consistentemente parásitos en sangre a una concentración 10 tripanosomas/ml. Durante la evaluación de campo, más pacientes dieron positivo por mAECT-cl (96.5%) que por mAECT-s (78.9%, Chi2=6.93, p=0.008) y examen de linfa (77.2%, Chi2=7.67, p=0.005). El número de parásitos por colector fue significativamente mayor (7.2 vs 2.6, p=0.001) cuando se recolectó la capa leucocitaria en vez de utilizar la sangre.

Conclusión:  El uso del protocolo mAECT-cl permitió mejoras significativas en el diagnóstico parasitológico de THA en Guinea, sin costes adicionales. Vale la pena probarlo en otras áreas endémicas para T.b. gambiense.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Human African trypanosomiasis (HAT) or sleeping sickness is a disease found in sub-Saharan Africa caused by two sub-species of trypanosome, Trypanosoma brucei (T.b.) rhodesiense and T.b. gambiense, with the latter causing over 90% of all cases (Simarro et al. 2008). The disease is classically characterized by an early haemolymphatic phase followed by a late meningoencephalitic phase leading to neurological disorders and death. Owing to the absence of specific clinical symptoms and to the fact that available treatments are either highly toxic and/or require long-term hospitalization in specialized health structures (Welburn et al. 2009); diagnostic tools are of crucial importance (WHO 1998).

Diagnosis of T.b. gambiense HAT usually relies on mass screening with the card agglutination test for trypanosomiasis (CATT). This test is highly sensitive but lacks in specificity. Thus, parasitological tests on body fluids (blood or lymph juice) are required to confirm HAT. Various techniques such as the capillary tube centrifugation (CTC, WOO), the quantitative buffy coat (QBC) and the mini-anion exchange centrifugation technique (mAECT) were elaborated to concentrate parasite in blood prior to microscopic examination to increase sensitivity of parasitological diagnosis (Chappuis et al. 2005). The mAECT has proved to be the most sensitive method (Büscher et al. 2009). In mAECT, trypanosomes are separated from 350 μl of blood by anion exchange chromatography on diethylaminoethyl cellulose (DEAE). Eluted trypanosomes are then concentrated by slow centrifugation followed by direct microscopic examination of the sediment in a transparent collector tube. The large volume of blood examined allows the detection of less than 50 trypanosomes/ml (Büscher et al. 2009). However, these field-applicable parasitological tools still suffer from limited sensitivity, therefore denying or delaying necessary treatment (Lutumba et al. 2005). PCR using specific DNA probes (Chappuis et al. 2005), loop-mediated isothermal amplification (Njiru et al. 2008), and more recently, nucleic acid based amplification with oligochromatography (Mugasa et al. 2009) provides a better sensitivity compared to parasitological methods. However, molecular diagnosis is expensive, difficult to implement under field conditions and has not yet been included in the diagnosis procedures of National Control Programs (NCP).

These observations led us to evaluate a modified mAECT protocol to increase further the sensitivity of the test. It simply consists of adding an additional parasite concentration step by centrifugation of the venous blood collection tube (5 ml). Upon centrifugation, trypanosomes are concentrated in the buffy coat layer, a principle that is already applied in the CTC (Chappuis et al. 2005) but with much smaller volumes of blood.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

mAECT protocols

All tests were performed with 5 ml of blood in heparinized vacutainers from either (i) serial parasite dilutions made in blood (experimental evaluation) or (ii) patients (field evaluation). From each vacutainer, 350 μl of blood was first removed and processed on mAECT columns (INRB-model) according to standard operating procedures (http://www.finddiagnostics.org/programs/hat/find_activities/maect_sop/index.html). The vacutainer was then centrifuged 5 min at 1500 × g. Removal of the buffy coat (the concentrated leucocyte layer) was done using a 1-ml pipette tip that was previously cut out with scissors (at 5 mm from its extremity). The tip was then placed at the centre of the vacutainer just above (1 mm) the white blood cell layer, and 350 μl of buffy coat were removed slowly without moving the pipette (a small fraction of plasma and of concentrated red blood cells are also removed during the operation). The collected buffy coat suspension was then processed on mAECT columns according to the same protocol as blood.

Experimental evaluation

Trypanosomes (MBONR1 stock, isolated in Guinea) were grown on BalbC mice and added to the blood of a healthy donor to obtain serial parasites concentration of 50, 10, 5 and 1 trypanosomes/ml. Five mililitres of parasitized blood was then transferred to n (number of replicates, see below) heparinized vacutainers and processed on to mAECT columns as described earlier. Collectors were then given an arbitrary code, and parasites were counted blindly by a skilled microscopist who did all the readings. The experiment was carried out during three consecutive days using the blood of the same donor each time to minimize the effect of blood donor-dependent variability. Blood parasite concentrations of (i) 50 (n = 5) and 10 (n = 5) trypanosomes/ml; (ii) 10 (n = 5) and 5 (n = 5) trypanosomes/ml and (iii) 5 (n = 5) and 1 (n = 5) trypanosomes/ml were tested on day 1, day 2 and day 3 respectively.

Field evaluation

The field evaluation of the mAECT-bc protocol was performed during a medical survey in the Dubreka area, the most active HAT focus in Guinea (Camara et al. 2005). Mass screening was performed with the CATT, and all individuals with a CATT dilution titre ≥1/8 (n = 96) were submitted to lymph node aspirate examination whenever swollen lymph nodes were present (n = 62) and mAECT was performed both with blood and buffy coat. All mAECT columns were set up and read by the same technician. Reading in this case was not done blindly.

Ethical aspects

The study protocol was approved by the Guinean Ministry of Health and the field survey was conducted under the supervision of the National Control Program. All patients diagnosed in the frame of this study were treated at the Dubreka treatment centre.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Experimental evaluation of the mAECT-bc protocol showed that the average number of trypanosomes recovered from mAECT-bc collectors were consistently and significantly higher than for mAECT-blood collectors, at all parasite concentrations tested (P = 0.005, P = 0.001 and P = 0.02 at 50, 10 and 5 trypanosome/ml respectively by the paired t-test) except for 1 trypanosome/ml, where both tests were negative (Figure 1). In agreement with previous reported sensitivity thresholds, all mAECT-blood collectors were positive at 50 trypanosomes/ml, while only 50% (5/10) and 30% (3/10) were positive at 10 and 5 trypanosomes/ml, respectively. In contrast, 100% (10/10) of mAECT-bc collectors were still positive at 10 trypanosomes/ml, and 70% (7/10) at 5 trypanosomes/ml. Thus, sensitivity of mAECT-bc was increased at least five fold considering the last concentration with 100% positivity as the threshold of the test.

image

Figure 1.  Experimental evaluation of mAECT-bc over mAECT-blood. Serial blood dilution of a Trypanosoma brucei gambiense strain isolated in Guinea (MBONR1) and produced on BalbC mice was used to compare mini anion exchange centrifugation test (mAECT) performed directly from blood or from buffy coat. Blood parasite concentrations tested were 50 trypanosomes/ml (n = 5), 10 trypanosomes/ml (n = 10), 5 trypanosomes/ml (n = 10) and 1 trypanosome/ml (n = 5). The graph represents the mean parasite counts of replicates, calculated for each parasite concentration. Standard error of the mean is also indicated. The proportions of positive collectors (with at least one trypanosome seen) are indicated in the table under the graph.

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The mAECT-bc protocol was then evaluated in the field during a medical survey carried out in the Dubreka HAT focus. A total of 57 individuals were diagnosed as patients with HAT during this survey and were positive to at least one of the performed parasitological tests (lymph examination, mAECT-blood or mAECT-bc). Thirty-seven patients (64.9%) were positive to all tests, 2 (3.5%) were positive to lymph juice examination only and 5 (8.7%) were positive to mAECT-bc only (Figure 2). More patients were positive to mAECT-bc (96.5%) than to lymph juice examination (77.2%, χ2 = 7.67, P = 0.005) and mAECT-blood (78.9%, χ2 = 6.93, P = 0.008). The mean number of trypanosomes counted in collectors (Figure 3) was significantly higher for mAECT-bc (7.2 [3.8–11.9] vs. 2.6 [1.7–3.5], P = 0.001 by the paired t-test). This represented a gain of 4.5 trypanosomes per collector, rendering parasitological investigations quicker, easier and more reliable in the case of negative results.

image

Figure 2.  Distribution of HAT cases according to the result of parasitological tests. Percentage of HAT cases (numbers are indicated above the histogram bars) is shown according to the three parasitological tests performed. For all subjects with CATT titration results ≥1/8, direct microscopic examination of lymph node aspirates was performed whenever cervical lymph nodes were present and 5 ml of venous blood was collected on a heparinized collection tube. Three hundred and fifty microlitres of blood or 350 μl of buffy coat (taken after centrifugation of the tube, 5 min at 1500 × g) was processed on mini anion exchange centrifugation test columns. Results are shown for all patients with HAT that were positive to at least one parasitological test (n = 57).

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image

Figure 3.  Comparison of trypanosomes counts in mAECT-blood and mAECT-bc collectors. Results are presented only for patients who were positive to at least one of the mini anion exchange centrifugation test (mAECT) protocols (55/57 patients). Furthermore, as an accurate estimation of parasite number is difficult to estimate when a large number of trypanosomes are recovered in collectors, we limited the statistical analysis to patients in whom parasite counts in mAECT-blood was <15 (50/55 patients). For parasite counts that were >15 in mAECT-blood collectors, parasite counts in mAECT-bc were usually too high to be accurately evaluated and thus were not included. The horizontal bars represent the mean of mAECT-bc parasite counts. The dotted line is the line y = x above which parasite counts were higher in mAECT-bc than in mAECT-blood.

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The mAECT-bc method was very much appreciated by the field team and it is now included in the HAT diagnosis chain instead of mAECT-blood by the National Control Program of Guinea. Furthermore, being easier to read, it enabled the diagnostic of 8.7% more patients, compared to the diagnosis protocol relying on examination of lymph nodes and mAECT-blood. We are convinced that the introduction of mAECT-bc in Guinea will represent a significant advance for the control of the disease, without any additional cost. Further evaluation of mAECT-bc at larger scales in other T.b. gambiense endemic areas should now be encouraged.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We are particularly grateful to the technicians of the HAT NCP of Guinea for their help. This work was supported by the Institut de Recherche pour le Développement, the French Ministry of Foreign Affairs (FSP/REFS Project, scholarship of Oumou Camara) and the World Health Organization.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  • Büscher P, Mumba Ngoyi D, Kaboré J et al. (2009) Improved Models of Mini Anion Exchange Centrifugation Technique (mAECT) and Modified Single Centrifugation (MSC) for sleeping sickness diagnosis and staging. PLoS Neglected Tropical Diseases 3, e471.
  • Camara M, Kaba D, KagbaDouno M, Sanon JR, Ouendeno FF & Solano P (2005) Human African trypanosomiasis in the mangrove forest in Guinea: epidemiological and clinical features in two adjacent outbreak areas. Médecine Tropicale 65, 155161.
  • Chappuis F, Loutan L, Simarro P, Lejon V & Büscher P (2005) Options for field diagnosis of Human African Trypanosomiasis. Clinical Microbiology Reviews 18, 133146.
  • Lutumba P, Robays J, Miaka C et al. (2005) The efficiency of different detection strategies of human African trypanosomiasis by T. b. gambiense. Tropical Medicine and International Health 10, 347356.
  • Mugasa CM, Laurent T, Schoone GJ, Kager PA, Lubega GW & Schallig HD (2009) Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples. Journal of Clinical Microbiology 47, 630635.
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  • Simarro PP, Jannin J & Cattand P (2008) Eliminating human African trypanosomiasis: where do we stand and what comes next? PLoS Medicine 5, e55.
  • Welburn SC, Maudlin I & Simarro PP (2009) Controlling sleeping sickness – a review. Parasitology 136, 19431949.
  • WHO (1998) Control and surveillance of African trypanosomiasis. World Health Organization Technical Report Series 881, 1113.