Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo
Author(s)
Patrick Mitashi1,
Pascal Lutumba1,
Crispin Lumbala2,
Paul Bessell3,
Sylvain Biéler3*,
Joseph Mathu Ndung’u3
Affiliation(s)
1 Tropical Medicine Department, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of the Congo. 2 Programme National de Lutte contre la Trypanosomiase Humaine Africaine (PNLTHA), Croisement de l’Avenue de la Libération et du Boulevard Triomphal 1, Kinshasa, Democratic Republic of the Congo. 3 Foundation for Innovative New Diagnostics (FIND), Campus Biotech, Chemin des Mines 9, Geneva, Switzerland.
1 Tropical Medicine Department, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of the Congo. 2 Programme National de Lutte contre la Trypanosomiase Humaine Africaine (PNLTHA), Croisement de l’Avenue de la Libération et du Boulevard Triomphal 1, Kinshasa, Democratic Republic of the Congo. 3 Foundation for Innovative New Diagnostics (FIND), Campus Biotech, Chemin des Mines 9, Geneva, Switzerland.
ABSTRACT
Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.
Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.
KEYWORDS
Human African Trypanosomiasis, Trypanosome, LED Fluorescence Microscopy, Red Blood Cell Lysis, Diagnosis
Human African Trypanosomiasis, Trypanosome, LED Fluorescence Microscopy, Red Blood Cell Lysis, Diagnosis
Cite this paper
Mitashi, P. , Lutumba, P. , Lumbala, C. , Bessell, P. , Biéler, S. and Ndung’u, J. (2015) Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo. Microscopy Research, 3, 17-25. doi: 10.4236/mr.2015.32003.
Mitashi, P. , Lutumba, P. , Lumbala, C. , Bessell, P. , Biéler, S. and Ndung’u, J. (2015) Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo. Microscopy Research, 3, 17-25. doi: 10.4236/mr.2015.32003.
References
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Clinical Infectious Diseases, 56, 195-203. [5] Kennedy, P.G. (2008) Diagnosing Central Nervous System Trypanosomiasis: Two Stage or Not to Stage? Transactions of the Royal Society of Tropical Medicine and Hygiene, 102, 306-307. [6] Kennedy, P.G. (2006) Diagnostic and Neuropathogenesis Issues in Human African Trypanosomiasis. International Journal for Parasitology, 36, 505-512. [7] Brun, R. and Blum, J. (2012) Human African Trypanosomiasis. Infectious Disease Clinics of North America, 26, 261- 273. [8] Lejon, V., Roger, I., Mumba, N.D., Menten, J., Robays, J., N’Siesi, F.X., Bisser, S., Boelaert, M. and Büscher, P. (2008) Novel Markers for Treatment Outcome in Late-Stage Trypanosoma brucei gambiense Trypanosomiasis. Clinical Infectious Diseases, 47, 15-22.
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http://dx.doi.org/10.1371/journal.pntd.0000737 [15] Ndung’u, J.M., Bieler, S. and Roscigno, G. (2010) “Piggy-Backing” on Diagnostic Platforms Brings Hope to Neglected Diseases: The Case of Sleeping Sickness. PLoS Neglected Tropical Diseases, 4, e715.
http://dx.doi.org/10.1371/journal.pntd.0000715 [16] Biéler, S., Matovu, E., Mitashi, P., Ssewannyana, E., Bi Shamamba, S.K., Bessell, P.R. and Ndung’u, J.M. (2012) Improved Detection of Trypanosoma brucei by Lysis of Red Blood Cells, Concentration and LED Fluorescence Microscopy. Acta Tropica, 121, 135-140.
http://dx.doi.org/10.1016/j.actatropica.2011.10.016 [17] Chappuis, F., Loutan, L., Simarro, P., Lejon, V. and Büscher, P. (2005) Options for Field Diagnosis of Human African Trypanosomiasis. Clinical Microbiology Reviews, 18, 133-146.
http://dx.doi.org/10.1128/CMR.18.1.133-146.2005 [18] Mitashi, P., Hasker, E., Lejon, V., Kande, V., Muyembe, J.J., Lutumba, P. and Boelaert, M. (2012) Human African Trypanosomiasis Diagnosis in First-Line Health Services of Endemic Countries, a Systematic Review. PLOS Neglected Tropical Diseases, 6, e1919. [19] Agresti, A. and Coul, B.A. (1998) Approximate Is Better than “Exact” for Interval Estimation of Binomial Proportions. American Statistician, 52, 119-126.
http://dx.doi.org/10.2307/2685469 [20] Crawley, M.J. (2007) The R Book. John Wiley & Sons, Chichester.
http://dx.doi.org/10.1002/9780470515075 [21] R Development Core Team (2008) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. [22] Hansen, D.W., Hunter, D.T., Richards, D.F. and Allred, L. (1970) Acridine Orange in the Staining of Blood Parasites. Journal of Parasitology, 56, 386-387.
http://dx.doi.org/10.2307/3277684 [23] Lejon, V., Ameel, V., Brandt, J., Van den Bossche, P. and Büscher, P. (2003) Development of a Direct Immunofluorescence Test (DIFAT) for Parasitological Diagnosis of African Trypanosomosis. In: Wgzo, J., Ed., International Scientific Council for Trypanosomiasis Research & Control (ISCTRC), Twenty Seventh Meeting, Pretoria, Organization of African Unity (OAU), Nairobi, Scientific and Technical Research Council (STRC), 2005, 185-189. [24] Lenz, D., Kremsner, P.G., Lell, B., Biallas, B., Boettcher, M., Mordmüller, B. and Adegnika, A.A. (2011) Assessment of LED Fluorescence Microscopy for the Diagnosis of Plasmodium falciparum Infections in Gabon. Malaria Journal, 10, 194.
http://dx.doi.org/10.1186/1475-2875-10-194
[1] WHO (2013) Control and Surveillance of Human African Trypanosomiasis. Report of a WHO Expert Committee. World Health Organization Technical Report Series, 984, 1-250. [2] Simarro, P.P., Diarra, A., Ruiz Postigo, J.A., Franco, J.R. and Jannin, J.G. (2011) The Human African Trypanosomiasis Control and Surveillance Programme of the World Health Organization 2000-2009: The Way Forward. PLoS Neglected Tropical Diseases, 5, e1007.
http://dx.doi.org/10.1371/journal.pntd.0001007 [3] WHO (2013) Trypanosomiasis, Human African (Sleeping Sickness).
http://www.who.int/mediacentre/factsheets/fs259/en/ [4] Alirol, E., Shrumpf, D., Heradi, J.A., Riedel, A., de, P.C., Quere, M. and Chappuis, F. (2013) Nifurtimox-Eflornithine Combination Therapy (NECT) for Second-Stage Gambiense Human African Trypanosomiasis: MSF Experience in the Democratic Republic of the Congo.
Clinical Infectious Diseases, 56, 195-203. [5] Kennedy, P.G. (2008) Diagnosing Central Nervous System Trypanosomiasis: Two Stage or Not to Stage? Transactions of the Royal Society of Tropical Medicine and Hygiene, 102, 306-307. [6] Kennedy, P.G. (2006) Diagnostic and Neuropathogenesis Issues in Human African Trypanosomiasis. International Journal for Parasitology, 36, 505-512. [7] Brun, R. and Blum, J. (2012) Human African Trypanosomiasis. Infectious Disease Clinics of North America, 26, 261- 273. [8] Lejon, V., Roger, I., Mumba, N.D., Menten, J., Robays, J., N’Siesi, F.X., Bisser, S., Boelaert, M. and Büscher, P. (2008) Novel Markers for Treatment Outcome in Late-Stage Trypanosoma brucei gambiense Trypanosomiasis. Clinical Infectious Diseases, 47, 15-22.
http://dx.doi.org/10.1086/588668 [9] Lejon, V. and Büscher, P. (2005) Review Article: Cerebrospinal Fluid in Human African Trypanosomiasis: A Key to Diagnosis, Therapeutic Decision and Post-Treatment Follow-Up. Tropical Medicine & International Health, 10, 395- 403. [10] Van Nieuwenhove, S., Betu-Ku-Mesu, V.K., Diabakana, P.M., Declercq, J. and Bilenge, C.M. (2001) Sleeping Sickness Resurgence in the DRC: The Past Decade. Tropical Medicine & International Health, 6, 335-341. [11] Simarro, P.P., Jannin, J. and Cattand, P. (2008) Eliminating Human African Trypanosomiasis: Where Do We Stand and What Comes Next? PLoS Medicine, 5, e55.
http://dx.doi.org/10.1371/journal.pmed.0050055 [12] Franco, J.R., Simarro, P.P., Diarra, A., Ruiz-Postigo, J.A. and Jannin, J.G. (2012) The Human African Trypanosomiasis Specimen Biobank: A Necessary Tool to Support Research of New Diagnostics. PLoS Neglected Tropical Diseases, 6, e1571.
http://dx.doi.org/10.1371/journal.pntd.0001571 [13] Büscher, P., Mumba, N.D., Kabore, J., Lejon, V., Robays, J., Jamonneau, V., Bebronne, N., Van der Veken, W. and Biéler, S. (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. http://dx.doi.org/10.1371/journal.pntd.0000471 [14] Matovu, E., Mugasa, C.M., Ekangu, R.A., Deborggraeve, S., Lubega, G.W., Laurent, T., Schoone, G.J., Schallig, H.D. and Büscher, P. (2010) Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligo- chromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda. PLoS Neglected Tropical Diseases, 4, e737.
http://dx.doi.org/10.1371/journal.pntd.0000737 [15] Ndung’u, J.M., Bieler, S. and Roscigno, G. (2010) “Piggy-Backing” on Diagnostic Platforms Brings Hope to Neglected Diseases: The Case of Sleeping Sickness. PLoS Neglected Tropical Diseases, 4, e715.
http://dx.doi.org/10.1371/journal.pntd.0000715 [16] Biéler, S., Matovu, E., Mitashi, P., Ssewannyana, E., Bi Shamamba, S.K., Bessell, P.R. and Ndung’u, J.M. (2012) Improved Detection of Trypanosoma brucei by Lysis of Red Blood Cells, Concentration and LED Fluorescence Microscopy. Acta Tropica, 121, 135-140.
http://dx.doi.org/10.1016/j.actatropica.2011.10.016 [17] Chappuis, F., Loutan, L., Simarro, P., Lejon, V. and Büscher, P. (2005) Options for Field Diagnosis of Human African Trypanosomiasis. Clinical Microbiology Reviews, 18, 133-146.
http://dx.doi.org/10.1128/CMR.18.1.133-146.2005 [18] Mitashi, P., Hasker, E., Lejon, V., Kande, V., Muyembe, J.J., Lutumba, P. and Boelaert, M. (2012) Human African Trypanosomiasis Diagnosis in First-Line Health Services of Endemic Countries, a Systematic Review. PLOS Neglected Tropical Diseases, 6, e1919. [19] Agresti, A. and Coul, B.A. (1998) Approximate Is Better than “Exact” for Interval Estimation of Binomial Proportions. American Statistician, 52, 119-126.
http://dx.doi.org/10.2307/2685469 [20] Crawley, M.J. (2007) The R Book. John Wiley & Sons, Chichester.
http://dx.doi.org/10.1002/9780470515075 [21] R Development Core Team (2008) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. [22] Hansen, D.W., Hunter, D.T., Richards, D.F. and Allred, L. (1970) Acridine Orange in the Staining of Blood Parasites. Journal of Parasitology, 56, 386-387.
http://dx.doi.org/10.2307/3277684 [23] Lejon, V., Ameel, V., Brandt, J., Van den Bossche, P. and Büscher, P. (2003) Development of a Direct Immunofluorescence Test (DIFAT) for Parasitological Diagnosis of African Trypanosomosis. In: Wgzo, J., Ed., International Scientific Council for Trypanosomiasis Research & Control (ISCTRC), Twenty Seventh Meeting, Pretoria, Organization of African Unity (OAU), Nairobi, Scientific and Technical Research Council (STRC), 2005, 185-189. [24] Lenz, D., Kremsner, P.G., Lell, B., Biallas, B., Boettcher, M., Mordmüller, B. and Adegnika, A.A. (2011) Assessment of LED Fluorescence Microscopy for the Diagnosis of Plasmodium falciparum Infections in Gabon. Malaria Journal, 10, 194.
http://dx.doi.org/10.1186/1475-2875-10-194