Parasite diversity and innovative serology: development of Trypanosoma cruzi lineage-specific ...

178 Pages·2015·14.41 MB·English

by  Tapan Bhattacharyya

LSHTM Research Online
Bhattacharyya, T; (2015) Parasite diversity and innovative serology: development of Try-
panosoma cruzi lineagespecific diagnosis of Chagas disease and of prognostic assays for vis-
ceral leishmaniasis. PhD thesis, London School of Hygiene & Tropical Medicine. DOI:
https://doi.org/10.17037/PUBS.02173663
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Parasite diversity and innovative serology: development of Trypanosoma 
cruzi lineage‐specific diagnosis of Chagas disease and of prognostic assays for 
visceral leishmaniasis. 

Tapan BHATTACHARYYA 

Thesis submitted in accordance with the requirements for the degree of  
Doctor of Philosophy of the University of London  

November 2014 

Department of Pathogen Molecular Biology 

Faculty of Infectious and Tropical Diseases 

LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE, 
UNIVERSITY OF LONDON 

Funded by European Commission Seventh Framework Programmes for Research: 
‘ChagasEpiNet’ and ‘NIDIAG’ 

Research group affiliation:   Professor Michael A. Miles 

Faculty of Infectious and Tropical Diseases
I, Tapan Bhattacharyya, confirm that the work presented in this thesis is my own.
Where information has been derived from other sources, I confirm that this has been
indicated in the thesis.

Tapan BHATTACHARYYA
November 2014

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ABSTRACT
Trypanosoma cruzi and the Leishmania donovani complex are parasitic protozoa
that, respectively, cause Chagas disease in the Americas, and visceral leishmaniasis,
predominantly in South Asia, East Africa, and Brazil.
T. cruzi is divided into the lineages TcI-TcVI. The relationship between infecting
lineage(s) and spectrum of clinical presentations remains poorly understood. This project
developed lineage-specific serology to identify an individual’s history of lineage infection. A
high level of polymorphism in the surface mucin TSSA was identified, and lineage-specific
synthetic peptides based on this diversity were applied here in ELISA with chagasic sera
from endemic countries. Peptide TSSApep-II/V/VI, based on a sequence common to those
lineages, was widely recognised by sera from Southern Cone countries, and also
unexpectedly by four samples from Ecuador; TSSApep-V/VI, which differs by a single amino
acid from TSSApep-II/V/VI, was also recognised in these regions. A single TSSApep-IV
reaction was seen in both Colombia and Venezuela. However, TSSApep-I was rarely and
weakly recognised among the serum panel. Among the Brazilian patients, a much higher
proportion of TSSApep-II/V/VI responders had ECG abnormailities than non-responders
(38% vs. 17%, p<0.0001).
Rapid diagnostic tests for L. donovani complex infection based on rK39 antigen have
lower sensitivity in East Africa compared to South Asia. The homologous sequences of rK39,
and of another proposed diagnostic antigen HASPB, were amplified from a panel of East
African L. donovani strains, and compared to published sequences, revealing significant
diversity from rK39 and South Asian sequences, and non-canonical combinations of HASPB
repeats. Cohorts of Indian and Sudanese VL patients were assayed by ELISA for anti-
Leishmania IgG levels. There was an overall 46.8 – 61.7 fold lower response in the Sudanese
cohort, as calculated by mean reciprocal log t titres, regardless of antigen source, patient
10 50
gender or age.
An investigation into the association of IgG subclass reactivity with VL clinical status
revealed significantly elevated IgG1 levels in patients with active (pre-treatment) VL and
those with post-therapy relapse compared to those deemed to be cured. A novel prototype
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rapid immunochromatographic test to detect IgG1 gave > 80% of relapsed VL patients as
IgG1 positive, and 80% of cured patients as IgG1 negative.
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ACKNOWLEDGEMENTS - General
The research presented in this thesis was supported by the EC Framework Programme
Projects ‘Comparative epidemiology of genetic lineages of Trypanosoma cruzi’
(ChagasEpiNet, co-ordinator Prof MA Miles, LSHTM), and ‘Syndromic approach to neglected
infectious diseases at primary health care level: an international collaboration on integrated
diagnosis-treatment platforms’ (NIDIAG, co-ordinator Prof M Boelaert, ITG).

In 2009, the centenary of the discoveries of Carlos Chagas, I had the great privilege to begin
my doctoral research with, and receive the mentoring of, Prof. Michael A. Miles, Faculty of
Infectious & Tropical Diseases, LSHTM. I have benefited greatly from expertise, reagents,
technologies, and support provided from many colleagues:
Miles group, LSHTM Nidia Acosta, Armon Ayandeh, Rania Baleela, Duncan Bowes, Andrew
Falconar, Sinead Fitzpatrick, Michael Gaunt, Marissa Gripenberg, Hannah Hafezi, Kiera
Hayes, Osama Idris, Michael Lewis, Martin Llewellyn, Tegwen Marlais, Isabel Mauricio,
Louisa Messenger, Emily Mills, Sara Silva Pereira, Trang Tran, Matthew Yeo.
ChagasEpiNet partners Prof Björn Andersson, Oscar Franzén (Karolinska Institutet, Sweden);
Christian Barnabé (GEMI-IDR, France); Prof Hernan Carrasco, Maikell Segovia (Universidad
Central de Venezuela); Patricio Diosque (Universidad Nacional de Salta, Argentina); Prof
Alejandro Luquetti, Suelene Tavares, Liliane Siriano, Christian Rodrigues, Gabi Miranda,
Rosângela Amaral (Hospital das Clinicas, Federal University of Goias, Brazil); Prof Ana
Jansen, Fabiana Rocha, Samanta Xavier, Juliana Barros, André Roque, André Perreira
(FIOCruz, Rio de Janeiro, Brazil); Prof Yves Carlier, Sabrina Cencig (Université Libre de
Bruxelles, Belgium); Prof Mario Grijalva, Jaime Costales (Pontificia Universidad Católica del
Ecuador); Prof Felipe Guhl, Juan-David Ramirez (Universidad de los Andes, Colombia); Lineth
Garcia (Bolivia).
NIDIAG partners Prof Marleen Boelaert (Instituut voor Tropische Geneeskunde, Belgium),
Prof Sayda El-Safi, Dr Osman Ahmed, Dr Osama Eisa, Dr A Saad (University of Khartoum,
Sudan); Prof Shyam Sundar, Om Prakash Singh, Rajiv Srivastava, Dinesh Prajapati (Banaras
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Hindu University, India); Pascal Mertens, Caroline Thunissen (Coris BioConcept, Belgium);
Prof Suman Rijal, Narayan Bhattarai, Keshav Rai (BP Koirala Institiue of Health Sciences,
Dharan, Nepal).
LSHTM Ozan Gundogdu, Eloise Thompson, Carolyn Stanley, Keith Flanders.
External Carolyn Hyde (University College London); Sergey Litvinov (Aptum Biologicals).

ACKNOWLEDGEMENTS – Specific contributions of others to the work described herein
Production of synthetic peptides, experimental design & analysis, comments/editing of
manuscripts: Andrew Falconar
Access to chagasic and control sera: Prof Alejandro Luquetti, Prof Mario Grijalva, Jaime
Costales, Prof Felipe Guhl, Juan-David Ramirez, Lineth Garcia, Prof Hernan Carrasco, Patricio
Diosque
Supply of T. cruzi strains and/or DNA: Michael Lewis, Louisa Messenger, Martin Llewellyn,
Cristian Barnabe
Assisted phylogenetic analyses of sequences generated by candidate: Louisa Messenger,
Michael Lewis, Matthew Yeo
Novel bioinformatic tools: Sergey Litvinov
Access to visceral leishmaniasis and control sera: Prof Shyam Sundar, Om Prakash Singh,
Rajiv Srivastava, Dinesh Prajapati, Prof Sayda El-Safi, Dr Osman Ahemd, Dr A Saad
Production of IgG1 immunochromatographic rapid diagnostic test: Pascal Mertens, Caroline
Thunissen
Students performing research work under the candidate’s supervision: Duncan Bowes,
Armon Ayandeh, Marissa Gripenberg, Trang Tran, Sara Silva Pereira.

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CONTENTS
TITLE PAGE “Parasite diversity and innovative serology: development of Trypanosoma cruzi lineage-
specific diagnosis of Chagas disease and of prognostic assays for visceral
leishmaniasis.”....................................................................................................................page 1
ABSTRACT..................................................................................................................................3
ACKNOWLEDGEMENTS.............................................................................................................5
CONTENTS.................................................................................................................................7
ABBREVIATIONS......................................................................................................................12
LIST OF TABLES........................................................................................................................15
LIST OF FIGURES......................................................................................................................16
1.GENERAL INTRODUCTION
1.1 Trypanosoma cruzi and Chagas disease............................................................................18
1.2 Leishmania donovani complex and visceral leishmaniasis ...............................................19
2. LITERATURE REVIEW
2.1 Carlos Chagas and the discovery of T. cruzi.......................................................................21
2.2 T. cruzi
2.2.1 Taxonomy...........................................................................................................21
2.2.2 Life cycle.............................................................................................................22
2.2.3 Ultrastructure.....................................................................................................23
2.2.4 Vertebrate host cell invasion..............................................................................24
2.2.5 Vectors...............................................................................................................25
2.2.6 Other routes of infection....................................................................................27
2.3 Chagas disease
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2.3.1 Clinical manifestations.......................................................................................28
2.3.2 Diagnosis............................................................................................................31
2.3.3 Prevalence..........................................................................................................34
2.3.4 Treatment and management.............................................................................35
2.4 Control programmes.........................................................................................................37
2.5 T. cruzi epidemiology and ecology....................................................................................38
2.6 T. cruzi reference genome sequencing..............................................................................44
2.7 Research needs: Chagas disease.......................................................................................45
2.8 Discovery of Leishmania. ..................................................................................................46
2.9 Leishmania spp.
2.9.1 Taxonomy...........................................................................................................47
2.9.2 Life cycle ............................................................................................................47
2.9.3 Vectors...............................................................................................................48
2.10 Visceral leishmaniasis
2.10.1 Clinical manifestations ....................................................................................50
2.10.2 Diagnosis..........................................................................................................52
2.10.3 Treatment and management...........................................................................56
2.11 Control strategies............................................................................................................59
2.12 L. donovani complex population structure.....................................................................59
2.13 Leishmania reference genome sequences.....................................................................63
2.14 Research needs: visceral leishmaniasis...........................................................................64
3. AIMS & OBJECTIVES
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3.1 T. cruzi and Chagas disease............................................................................................65
3.2 L. donovani complex and visceral leishmaniasis...............................................................65
4. MATERIALS & METHODS
4.1 Parasite strains..................................................................................................................67
4.2 Oligonucletide sequences.................................................................................................67
4.3 PCR, DNA electrophoresis and cycle sequencing..............................................................68
4.4 Peptide synthesis..............................................................................................................69
4.5 Sources of human samples................................................................................................70
4.6 ELISA..................................................................................................................................71
5. PUBLICATIONS ARISING......................................................................................................72
T. cruzi and Chagas disease
ANNEX 1: Bhattacharyya T, Brooks J, Yeo M, Carrasco HJ, Lewis MD, Llewellyn MS, Miles MA
(2010). Analysis of molecular diversity of the Trypanosoma cruzi trypomastigote small
surface antigen reveals novel epitopes, evidence of positive selection and potential
implications for lineage-specific serology. Int J Parasitol 40 921-
928..........................................................................................................................................74

ANNEX 2: Bhattacharyya T, Falconar A, Luquetti A, Cosatles J, Grijalva M, Lewis M,
Messenger L, Tran T, Ramirez JD, Guhl F, Carrasco H, Diosque P, Litvinov S, Miles MA (2014).
Development of peptide-based lineage-specific serology for chronic Chagas disease:
geographical and clinical distribution of epitope recognition. PLoS Negl Trop Dis 8
e2892......................................................................................................................................76
L. donovani complex and visceral leishmanisis
ANNEX 3: Bhattacharyya T, Boelaert M, Miles MA (2013). Comparison of visceral
leishmaniasis diagnostic antigens in African and Asian Leishmania donovani reveals
extensive diversity and continent-specific polymorphisms. PLoS Negl Trop Dis 7
e2057......................................................................................................................................78
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