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GUINEA  WORM:
SOCIO -CULTURAL STUDIES , MORPHOMETRY, 
HISTOMORPHOLOGY, VECTOR  SPECIES AND 
DNA PROBE FOR DRACUNCULUS  SPECIES.
A thesis Presented to the Board o f  Graduate Studies, 
University o f  Ghana, Legon.
Ghana.
In fulfillment o f  the 
Requirements for the Degree 
o f  Doctor o f  Philosophy (Ph.D.)
(Zoology).
Langbong Bimi 
M. Phil.
Department o f  Zoology,
Faculty o f  Science,
University o f  Ghana 
Legon, Accra, Ghana.
September 2001
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DECLARATION
I do hereby declare that except for references to other people’s investigations which I 
duly acknowledged, this exercise is the result o f  my own original research, and that this 
thesis, either in whole, or in part, has not been presented for another degree elsewhere.
PRINCIPAL INVESTIGATOR SIGNATURE DATE
LANGBONG BIMI -==4=^" .
(STUDENT)
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1This thesis is dedicated to the Bimi family in memory o f  
our late father -  Mr. Combian Bimi Dapaah (CBD), 
affectionately known as Nanaanbuang  by his admirers.
You never limited us 
with any preconceived notions 
o f  what we could or could not achieve.
DEDICATION
You made us to understand that the best kind o f  knowledge to have is that which  
is learned for its own sake, and that even the largest task can be accomplished i f  it 
is done one step at a time.
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SUPERVISORS
1.
2 .
3.
6 .
J,
NATU
4.
DR. JOHN KPIKPI
Department o f  Zoology 
University o f  Ghana, Legon 
Ghana.
DR. DAVID WILSON
(HEAD, Department o f  Zoology) 
University o f  Ghana, Legon 
Ghana.
DR. DOMINIC EDOH
Department o f  Zoology 
University  o f  Ghana, Legon 
Ghana.
DR. (MRS.) MATILDA PAPPOE
School o f  Public Health 
University o f  Ghana, Legon 
Ghana
DATE
5. DR. NORMAN PIENIAZEK
Centers for D isease Control and Prevention 
Department o f  Parasitic D iseases 
Biology and Diagnostics Branch 
A tlanta -  USA
DR. MARK L. EBERHARD
(CHIEF, Biology and D iagnostics Branch) 
Department o f  Parasitic D iseases 
Centers for D isease Control and Prevention 
A tlanta - USA
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ACKNOWLEDGMENT
I would like to thank the many people who supported and helped me with this 
work. First I would like to express gratitude to my supervisor Dr. J. E. K. Kpikpi 
whose support and encouragement was instrumental in the development and 
execution o f  this research. I would also like to thank my committee members: Dr. 
(Mrs.) Matilda Pappoe who provided much help with the questionnaire design and 
interpretations, and Dr. Dominic Edoh for his review o f  the project materials, 
many helpful comments and for the critical discussions and inputs on the topic. 
To Dr. David Wilson, my thesis chair and Head o f  Department; I cannot thank 
you enough for your advice and guidance and for your w illingness to support me 
when 1 had to travel to the Centers for Disease Control and Prevention (CDC), 
Atlanta, to complete the project. You helped make the Ph.D. journey more 
enjoyable.
From early August 2000 until late August o f  2001, I had to carry out the 
molecular biology aspect o f  the project at the Centers for D isease Control and 
Prevention. Many people generously extended their hospitality to me, and I left 
with wonderful memories. I would like to acknowledge the unwavering support o f  
Dr. Alexandre J. DaSilva and Mrs. Susan B. Slemenda. To Ms. Maniphet V. 
Xayavong and Dr. laci N. Moura, I say God richly bless you for all the technical 
assistance, enthusiasm, help and support.
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For handling my paperwork and administrative needs, I cannot but simply say 
Jennifer W. Dickerson and Brenda Lester are just wonderful. Thanks for all the 
patience and understanding.
1 am grateful for the support and advice that 1 received from Dr. Norman J. 
Pieniazek, who had to “dream out” all the expertise I needed in the execution o f
the project. He had to also contend with my never-ending questions and 
clarifications. Your support and words o f  encouragement kept me sane. Thanks 
for all o f  your insightful feedback and words o f  encouragement. I would also like 
to acknowledge the immense contributions by Amanda Freeman, Mark L. 
Eberhard and Norman J. Pieniazek for getting the first sequence data on 
Dracunculus medinensis and Dracunculus insignis for the project.
My biggest personal thanks go to Dr. Daniel Colley (Director, Department o f  
Parasitic D iseases o f  the CDC), Dr. Ernesto Ruiz-Tiben (Technical Director, 
Global 2000 Guinea Worm Eradication Program), and Dr. Mark L. Eberhard 
(Chief, Biology and Diagnostics Branch - DPD o f  CDC), for securing funding for 
the molecular studies o f  the project. You gave me the opportunity to jump into a 
new field with a rare chance to do real science. Not everyone would give such a 
chance to someone so new to molecular biology. 1 know it was not easy keeping 
me at the CDC for the 12 months, but you did it. 1 simply say --I love you all.
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VPREFACE
This thesis is composed o f  eight main sections prefaced by an introduction and 
followed by an abstract o f  conclusions and recommendations. The first two o f  the 
main sections provide relevant background information about dracunculiasis 
(iChapter 1) and a literature review (Chapter 2). Chapter 3 presents a study o f  the 
socio-cultural practices o f  the local people and their belief systems that could aid 
in the transmission and sustenance o f  the disease. Chapters 4 and 5 present the 
morphometry and histomorphometry o f  the parasite respectively. Chapter 6 is an 
evaluation o f  the infection potentials o f  the various copepods found in the study 
area that could serve as vectors o f  the disease. A  DNA probe for Dracunculus 
species is presented in Chapter 7. Finally, an overall discussion o f  all the aspects 
o f  the research is presented in Chapter 8. It is anticipated that material from all the 
chapters will be submitted for publication.
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TABLE OF CONTENTS
Page
Dedication............................................................................   1
Acknowledgements..................................................................................................................... ...11
Preface...................................................................................................................................  v
Table o f contents............................................................................................................................v '
ABSTRACT................................................................................................................................—  1
CHAPTER! ............................................................................................................................  3
1.0 INTRODUCTION ............................................................................................  3
1.1 JUSTIFICATION OF STU DY ................................................................................7
1.2 SPECIFIC STUDY OBJECTIVES....................................................................... 9
1.3 DEFINITIONS...........................................................................................................10
CHAPTER 2 ....................................................................................................................................15
2.0 LITERATURE REVIEW .............................................................................................15
2.1 HISTORY AND IMPORTANT DATES OF DRACUNCULIASIS ................. 15
2.1.1 H istory ............................................................................................................................15
2.1.2 Important D a te s ...........................................................................................................16
2.2 EPIDEMIOLOGY AND TRANSM ISION ............................................................... 17
2.3 ETIOLOGY AND LIFE-CYCLE OF DRACUNCULUS MEDINENSIS.......... 18
2.4 CLINICAL MANIFESTATIONS AND PATHOLOGY.......................................23
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2.5 DIAGNOSIS..............................................................................................................................z
2.5.1 Clinical and Parasitological D iagnosis ............................................................................ 24
2.5.2 Immunological d iagnosis ..................................................................................................... 26
2.6 TREATMENT......................................................................................................................... 26
2.6.1 Surgery ......................................................................................................................................26
2.6.2 Chemotherapy ..............................................................................................................27
2.7 SOCIAL AND ECONOMIC EFFECTS........................................................................29
2.7.1 Cultural and Traditional B e lie fs .........................................................................................30
2.7.2 Socio-Economic Aspects o f  Dracunculiasis.................................................................. 31
2.8 CONTROL MEASURES.................................................................................................. 32
2.9 THE PARASITE.................................................................................................................. 34
2.9.1 M orphology............................................................................................................................34
2.9.2 Taxonomy............................................................................................................................... 36
2.10 THE VECTORS OF DRACUNCULUS MEDINENSIS...........................................38
2.11 ZOONOSIS............................................................................................................................ 43
2.12 GUINEA WORM ERADICATION PROGRAMMES (GW EPS).....................44
2.12.1 Global Perspective...............................................................................................................44
2.12.2 The Guinea Worm Eradication Programme in Ghana..............................................46
CHAPTER 3 ................................................................................................................................ ..
3.0 SOCIO-CULTULRAL STUDIES ............................................................................ 50
3.1 MATERIALS AND METHODS.................................................................................... 50
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3.1.1 Study Locale .............................................................................................................. ^
3.1.2 Retrospective Studies for Disease Prevalence..................................................51
3.2 RESULTS.....................................................................................................................................52
3.2.1 Prevalence o f  Guinea Worm Disease ................................................................ 52
3.2.2 Seasonal Distribution o f  Guinea Worm D ise a se .............................................52
3.2.3 Occupational Distribution o f  Guinea Worm D ise a se ....................................53
3.2.4 Perceptions o f  D isease Causation.........................................................................53
3.2.5 Perceptions o f  D isease Transm ission .................................................................. 54
3.2.6 Perceptions on D isease Curability........................................................................54
3.2.7 Perceptions on D isease Prevention ...................................................................... 55
3.2.8 Source o f  K now ledge............................................................................................... 55
3.2.9 Respondents’ Principal Sources o f  W ater.........................................................56
3.2.10 Respondents’ Alternative Sources o f  W ater.....................................................56
3.2.11 Common Activities At Pond/Dam S ite s ............................................................ 57
3.2.12 Socio-demographic correlates o f  dracunculiasis............................................ 58
3.3 D ISCUSSION .................................................................................................................. 61
CHAPTER 4 ......................................................................................................................................... 64
4 0 INVESTIGATION OF WORM  M ORPHOM ETRY ............................ 64
4.1 MATERIALS AND METHODS........................................................................64
4.1.1 Determination o f  Worm Length .............................................................64
4.1.2 Determination o f  Worm W eigh t............................................................65
4.1.3 Determination o f  Worm D iam eter........................................................65
viii
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4.2 RESULTS...................................................................................................................... 00
4.3 DISCUSSIONS AND CONCLUSIONS............................................................. 68
CHAPTER  ...................................................................................................................................70
5.0 HISTOMORPHOLOGY OF DRACUNCULUS MEDINENSIS 70
5.1 MATERIALS AND  METHODS............................................................. 70
5.1.1 Tissue Preparation......................................................................................... 70
5.2 RESULTS...................................................................................................................... 72
5.2.1 The Cephalic R eg ion .................................................................................72
5.2.2 The Pharyngeal R eg ion ............................................................................73
5.2.3 The M id-Section ........................................................................................73
5.2.4 The Posterior S ection .............................................................................. 74
5.3 DISCUSSIONS AND CONCLUSIONS..........................................................75
CHAPTER 6 ......................................................................................................................................... 76
6.0 VECTOR SPECIES OF DRACUNCULUS MEDINENSIS ................................ 76
6.1 MATERIALS AND  METHODS...............................................................76
6.1.1 Sampling ........................................................................................ 76
6.1.2 Identification.................................................................................. 76
6.1.3 Colony Maintenance....................................................................77
6.1.4 Evaluation o f  Infection Potentia ls........................................... 78
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X6.2.1 Copepod S p ec ie s .........................................................................................................
6.2.2 Infection Potentials..................................................................................................... 80
6.3 D ISCUSSION ..................................................................................................................... 82
CHAPTER 7 ...........................................................................................................................................85
7.0 MOLECULAR EPIDEM IOLOGY OF GUINEA  W O RM .................... 85
7.1 MATERIALS AND  METHODS............................................................................ 88
7.1.1 Worm C ollection ............................................................................88
7.1.2 DNA Extraction and Preparation..............................................88
7.1.3 Polymerase Chain Reaction (PC R ).......................................... 89
7.1.4 Verification o f  PCR Product...................................................... 90
7.1.5 DNA Sequencing ...........................................................................90
7.2 RESULTS..............................................................................................................92
7.3 CONCLUSIONS................................................................................................ 94
CHAPTER 8 ......................................................................................................................................... 95
8.0 GENERAL D ISCUSSION ....................................................................................... 95
REFERENCES ..................................................................................................................................... 139
APPENDIX A : QUESTIONNAIRE...........................................................................................  149
APPENDIX B: WORM MORPHOMETRY...............................................................................153
APPENDIX C: Bio 101 FastDNA K it ...........................................................................................154
APPENDIX D: QLAquick-Spin: PCR Purification K it ........................................................... 156
APPENDIX E: StrataPrep PCR PURIFICATION....................................................................158
6.2 RESULTS ...............................................................................................................................
80
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APPENDIX F: CENTRI-SEP PURIFICATION PROTOCOL 
APPENDIX G: Base Sequences o f  Dracunculus Isolates
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LIST OF TABLES
TABLE 1: WORM MORPHOMETRY.................................................................102
TABLE 2: CYCLOPOID COPEPODS FOUND IN THE STUDY AREA . 103
TABLE 3: INFECTIVITY STUDIES WITH ADULT COPEPODS.................... 104
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LIST OF FIGURES
FIGURE 1: LIFE CYCLE OF DRACUNCULUS MEDINENSIS ........................................105
FIGURE 2: A COPEPOD ............................................................................................................... 106
FIGURE 3: MAP OF GHANA ......................................................................................................... 107
FIGURE 4: MONTHLY DISTRIBUTION OF GUINEA WORM DISEASE..................108
FIGURE 5: OCCUPATIONAL DISTRIBUTION OF DRACUNCULIASIS............  109
FIGURE 6: PERCEPTIONS OF THE CAUSES OF GUINEA W ORM ......................... 110
FIGURE 7: PERCEPTIONS OF METHOD OF TRANSMISSION OF GUINEA WORM I l l
FIGURE 8: PERCEPTIONS ON HOW TO CURE INFECTION........................................112
FIGURE 9: PERCEPTIONS ON DISEASE PREVENTION ..............................................  113
FIGURE 10: SOURCES OF KNOWLEDGE PREVENTION............................................... 114
FIGURE 11: PRINCIPAL SOURCES OF W ATER ................................................................. 115
FIGURE 12: ALTERNATIVE SOURCES OF WATER TO PO ND S ............................... 116
FIGURE 13: COMMON ACTIVITIES AT POND S ITE ...................................................... 117
FIGURE 14: CEPHALIC REGION OF GUINEA W ORM ..................................................118
FIGURE 15: THE PHARYNGEAL REGION ..........................................................................119
FIGURE 16: ENLARGED VERSIONS...................................................................................... 120
FIGURE 17: MID-ANTERIOR REGION ................................................................................. 121
FIGURE 18: M ID-SECTION........................................................................................................ 122
FIGURE 19: MID-SECTION PRESENTING THE MUSCULATURE...........................123
FIGURE 20: LOWER M ID-SECTION....................................................................................... 124
FIGURE 21: THE EXTREME POSTERIOR REGION OF GUINEA W ORM  125
FIGURE 22: FIRST STAGE LARVA (L ,) OF GUINEA WORM........................................ 126
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FIGURE 23: THIRD STAGE LARVA (L3) OF GUINEA W ORM .................................... 127
FIGURES 24 - 28: CLINICAL PRESENTATIONS..............................................................  128
FIGURE 29: PHYLOGENETIC TREE FOR SMALL SUBUNIT RIBOSOMAL RNA  
SEQUENCES OF DRACUNCULUS MEDINENSIS, D. INSIGNIS, AND
SELECTED NEMA TODA...................................................................................... 133
FIGURE 30: DIAGRAM OF THE RIBOSOMAL DNA G ENE .......................................... 134
FIGURE 31: WORLD MAP SHOWING ORIGIN OF SPECIMENS.................................135
FIGURES 32 -  34: VERIFICATION OF PCR PRODUCTS............................................... 136
xiv
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Abstract
Dracunculiasis still remains as a disease o f  public health importance in Ghana, especially in 
the Northern, Volta and Brong-Ahafo Regions, despite over a decade o f  an Eradication 
Programme. In this study, attempts have been made to determ ine the problems associated 
with the eradication o f  Guinea worm in the Northern Region o f  Ghana, and suggestions 
provided to overcome them. Also investigated were the longitudinal anatomical variation o f  
the parasite, the vector species and a DNA probe for Dracunculus species. It was found out 
that 43% o f  the populace still got afflicted with the malady during the last transm ission 
season, with 33% suffering from the disease for the second consecutive year. The disease 
appears to be sustained by the very long transm ission season, spanning over six months. The 
knowledge index o f  the local people with respect to the disease was quite high. Over 50% o f 
respondents seem to be aware o f  disease causation, prevention, and management. 
Unfortunately, however, apathy, lack o f  motivation, and w'orker fatigue on the part o f  G WEP 
staff appear to be factors in the resurgence o f  the disease.
In the histomorphological part o f  the study, the longitudinal anatomical variation o f  the 
parasite was evaluated and its histomorpholgy illustrated. At maturity, the gut o f  the female 
guinea worm was found to be completely atrophied and the entire worm made-up o f  the 
larvae-filled uterus. Differences in the musculature from the anterior region, w ith much 
thicker muscles to the mid region were observed. The most important vectors o f  
dracunculiasis in the study areas are: M. keiferi —> M. aspericornis —> T. incisus —> T. 
inopinus —> T. oblongatus .
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1To probe for molecular epidemiology o f  Dracunculus species, DNA sequence isolates from a 
number o f  Guinea worms originating from 9 African countries, Pakistan and Yemen were 
compared with a previously assembled isolate, 1819bp (not yet submitted to the Gene Bank) 
from Uganda. The 18S nuclear small subunit ribosomal RNA (SSU-rRNA) gene o f  Guinea 
worm from human and canine cases o f  dracunculiasis in Africa were isolated by PCR and 
sequenced. Phylogenetic analysis (o f these sequences) and comparison with the sequences 
from Uganda revealed that Dracunculus isolates from human cases were indistinguishable 
from those o f  the dog case. These results certainly imply that incidental infections o f  Guinea 
worm could occur in certain domestic animals, and therefore, suggest that some domestic 
animals may serve as reservoirs for human Guinea worm infection.
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3At the dawn o f  the 21s' Century, the terrible trinity o f  poverty, ignorance and disease, 
unfortunately still plagues most developing countries. Among these is Dracunculiasis or 
Guinea worm disease. This malady belongs to a group o f  diseases generally known as 
Tropical Diseases, based on their geographical occurrence in the tropics. Guinea worm is not 
a cause o f  mortality, but it can be a real burden in terms o f  morbidity and suffering for those 
affected. Dracunculiasis was estimated to affect several million people annually at the close 
o f  the 1970s. However, the international water decade (1981-1990), resulted in global decline 
in incidence by 1996 to less than 153, 000 cases worldwide, 78% o f  which were reported 
from Sudan (Kale, 1990). The disease is still found among the poorest rural communities in 
areas without safe water supplies in Sub-Saharan Africa and the Arabian Peninsula. 
Gradually wom down by penury, these underprivileged people find themselves trapped in a 
vicious circle o f  poverty and disease (Richards and Hopkins. 1989).
Dracunculiasis is caused by a long, string-like, female worm (Dracunculus medinensis). Its 
larval form infects an intermediate crustacean host (copepods. water fleas) that commonly 
infests shallow ponds or step wells used as sources o f  drinking water. Known to cause human 
suffering since ancient times, the infection was referred to by physicians as early as the 
Graeco-Roman era and by Arab physicians in medieval times. Common names include 
Guinea worm and Medina worm. Linnaeus classified the worm in the eighteenth century.
Chapter 1
1.0 INTRODUCTION
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4-
Fedchenko, a Russian naturalist, described the life cycle in 1869-the  first time an 
invertebrate (arthropod) intermediate host was described for any parasitic disease o f  man.
This round worm is the largest o f  the tissue parasites affecting humans. The adult female, 
which carries from 1 to 3 million embryos, can measure up to 1 meter in length and 2 mm in 
diameter. The parasite migrates through the victim 's body causing severe pain, especially in 
the areas around the joints. The worm eventually emerges (from the feet in 90% o f  cases), 
causing an intensely painful oedema, a blister and then an ulcer. When the worm perforates 
the skin, intolerable pain is accompanied by fever, nausea and vomiting (Muller, 1971).
Dracunculiasis (less commonly, dracontiasis, and rarely, dracunculosis) is contracted by 
drinking contaminated water. The offending parasite, historically the scourge o f  Islamic 
pilgrimage, is aptly called the Little Dragon o f  Medina or scientifically Dracunculus 
medinensis (Linnaeus, 1758; as cited in Macpherson. 1981).
From the middle ages through to the 18th century there were many varying opinions as to the 
nature o f  the "fiery serpents" - believed to be anything from exposed nerves to dead tissue. It 
was the celebrated Swedish naturalist, Carolus Linnaeus who first suggested that they were 
in fact worms. In 1870, Alexei P. Fedchenko became aware o f  the life-cycle o f  Dracunculus 
medinensis and identified copepods as its intermediate host. By the end o f  the 19th century, 
scientists had become well aware o f  how the disease was transmitted.
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Dracunculiasis still represents a serious health risk for several millions o f  rural villagers in 
parts o f  Africa, the Middle East, and India. It affects only the rural poor who lack safe 
sources o f  drinking water for their households and places o f  work (agricultural plots).
Unlike most communicable diseases in developing countries, the greatest morbidity from 
Dracunculiasis occurs in adults. This may be the reason it has received less attention than 
those illnesses resulting in high morbidity and mortality in children. Because peak case rates 
often coincide with such agricultural activities as clearing land, planting, and harvesting, the 
disease is a major cause o f  agricultural work loss in many areas. Infected individuals are 
often crippled or disabled for many weeks each year from painful ulcers produced by the 
worms' emergence and complications resulting from secondary bacterial infections.
Dracunculiasis in Ghana declined substantially during 1992, the 3rd consecutive year o f  
control. A total o f 33,464 cases o f  Dracunculiasis were reported in 3 .185 villages, compared 
with 66,697 in 3,718 villages in 1991 (a decline o f  49.6%) and 179.556 in 6.873 villages (a 
decline o f  81.4%) in 1989. In addition, when compared with 1991. the percentage reduction 
in cases reportedly by month increased from 20.1% in January to 34% in March. 59.9%  in 
June, 81% in September, and 55.6% in December. During 1992. at least 84% o f the villages 
affected reported surveillance findings to national authorities on time each month (within 
20-30 days after the end o f  the reporting month), compared with 61% during 1991. Not 
withstanding the political will and massive international support, there is unfortunately an
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6apparent relapse and worker fatigue in the control efforts. This may account for resurgence o f 
the disease in some communities (Annual Report, GWEP, Northern Region, 1997).
Increases in reported cases o f  Guinea worm in the country called for the intensification o f  
efforts for the disease eradication. Thus, the Eradication Programme has been re-launched in 
a number o f endemic communities. Notably, the Programme had to be re-launched in 
Gusheigu (in the Gushiegu-Karaga District o f  the Northern Region) in 1998 by the President 
o f  Ghana. Also, in Wusuta (Volta Region), and Asenyensua (Eastern Region), the GWEP 
had to re-launch the programme in 1999 due to re-emergence o f  the disease, and again at 
Chamba in the Nanumba district in 2001.
In July 1999, the Northern Region was still leading in the number o f  cases in the country. In 
the first quarter o f  the year, the region recorded 2,370 cases o f  Guinea worm. The Tamale 
Municipality recorded the highest number o f  500 cases during the period. This was 
unfortunately, against the background o f  the government’s commitment towards eradicating 
Guinea worm in the country by the year 2000.
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Infection with Dracunculus medinensis is still widespread in the Northern Region o f  Ghana, 
where rural people drink from unprotected water sources such as ponds and small water 
catchments. The fact that the disease still exists in the country beyond the target date o f  
global eradication warrants further investigations. The importance o f  investigating the socio­
cultural factors that tend to sustain and maintain the disease transm ission despite a decade o f  
the Ghana Guinea Worm Eradication Programme (GGWEP) can therefore not be over­
emphasized.
Understanding the local perceptions o f  disease causation and treatment o f  Guinea worm, 
morbidity and local knowledge o f  transm ission and prevention is particularly crucial, 
especially those that might contribute to the persistence o f  the disease. Also, it would be 
helpful to know whether certain beliefs were more likely to contribute to Guinea worm 
infection. The level o f  acceptance o f  the interventions aimed at the disease such as the use o f 
filters, chemical pond treatment, and community organization to dig and maintain 
wells/dams has to be evaluated. Answers to these issues would provide insights into how to 
reinvigorate eradication efforts in Ghana, which remains one o f  three most highly endemic 
countries for Guinea worm (the other 2 being Nigeria and Sudan).
Furthermore, recent advances in copepod systematics have refined the level o f  taxonomic 
resolution of these freshwater copepods and it is now known that Mesocyclops leukarti does
f
1.1 JUSTIFICATION OF STUDY
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not occur in either Africa or India (Keifer, 1981; Van de Velde, 1984). 1 here is, therefore, an 
obvious need to record these taxonomic changes, review earlier records, and update the 
nomenclature o f  the hosts where possible.
Also, the correct identification o f  the copepod intermediate hosts is important in mapping out 
the geographical distribution and spread o f  the disease. This is vital in the development o f  
eradication programmes, which aim to combat the disease by vector control.
Lastly, since most o f  the key features used as morphological markers for systematic analysis 
o f  Dracunculus spp. are present only on the male worms, which are rarely ever available for 
study, there is the need to develop DNA probes for Dracunculus spp. for molecular 
characterization.
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1.2 SPECIFIC STUDY OBJECTIVES
The specific objective o f  the project were to:
1. Evaluate the socio-cultural practices and beliefs o f  the local people that seem to 
maintain and sustain the guinea worm disease in northern Ghana.
2. Ascertain the morphometric correlates o f  D. medinensis in relation to the sex o f 
patients.
3. Study the histomorphology o f  the female guinea worm
4. Document the true vectors o f  guinea worm in Northern Ghana.
5. Develop a simple and rapid molecular assay to determine if  a dracunculid worm 
extracted from man in an area under control is D. medinensis or another species.
6. Ascertain possible species types o f  the parasite
7. Evaluate the zoonotic aspects o f  dracunculiasis for reservoir hosts
8. The complete gene coding o f  all possible species and sub-species ( if  any)
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10
Despite the relative simp] icity o f  the life cycle and epidemiology o f  Dracunculus medinensis, 
some traditional terms need to be defined more precisely to promote communication and 
enhance the comparability o f  data over time and among different areas. The definitions that 
follow are based on current parasitologic and epidemiologic knowledge o f  the disease. As 
future research and experience adds to the understanding o f  dracunculiasis, these definitions 
may need revision. (Names and parts o f  the definitions in brackets indicate synonyms or 
optional inclusions).
1. Active case
A person in whom an investigator, health worker, or other trained person sees the 
Dracunculus medinensis worm beneath or extending from the skin. Also, a person w ith an 
acute skin lesion from which the larvae o f  D. medinensis have been identified by a trained 
person using microscopy or other means.
2. Presumptive case
[Retrospective or historical case] A person who reports having experienced the emergence o f  
one or more D. medinensis worms within the past 2 years.
1.3 DEFINITIONS
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3. Pre-patent case
A person who has been infected with dracunculiasis or who has ingested infective larvae but 
who has not yet manifested clinical symptoms or signs o f  the disease such as blistering, 
laceration, and worm emergence. There is no specific diagnostic test to identify such cases at 
present.
4. Affected community
A local administrative or social unit (e.g., village, hamlet, town, city) in which indigenous 
active or presumptive cases o f  dracunculiasis, or both, have been reported during the 
previous 2 years.
5. Incubation period
The period o f  time between ingestion o f  infective D. medinensis larvae by a person and the 
onset o f clinical symptoms.
6. Peak patency period
[French: period de mise en evidence communitaire] Period o f  the year during which more 
than 50 percent o f  all eases (in a community or defined geographic area) are reported. This 
definition applies to areas where incidence is seasonal.
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7. Individual patency period
[French: period de mise en evidence individual] The interval in an individual case between 
the time o f first parasitologic evidence o f  the worm beneath or extending from the skin (or 
onset o f  typical skin blister from which a worm will soon emerge) and the time o f  complete 
expulsion or extraction o f  the worm from the body.
8. Unprotected water source
Source o f drinking water that contains copepods and that either allows partial or total 
immersion o f  an infected person in the water source or permits water runoff to enter. Such 
sources are also likely to contain unacceptable levels o f  microbiological contaminants.
9. Protected water source
Source o f drinking water that prohibits the partial or total immersion o f  an active guinea 
worm case and contamination from ground runoff. Such sources are usually constructed so as 
to remain free from fecal pollution.
10. Infested water source
Source o f  drinking water containing copepod species capable o f  ingesting first-stage larvae 
(L,) o f  D. medinensis.
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11. Infective water source
Source o f drinking water containing copepods infected with third-stage larvae (L3) o f  D. 
medinensis.
12. Surveillance
The continuing collection, analysis, and feedback o f  epidemiologic data based on reporting 
or detection o f cases o f disease.
13. Passive case reporting
Recording o f  cases that voluntarily come to the attention o f  public health authorities. Passive 
reporting or surveillance is achieved through the establishment o f  routine reporting 
mechanisms and is useful for obtaining initial data on the distribution o f  dracunculiasis in an 
area.
14. Active case detection
Search for cases by representatives o f  the health system through a village-to-village or 
house-to-house survey or a sample o f  the population thought to be at risk. This method 
usually reveals more cases than does passive reporting.
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15. Incidence
The rate o f  appearance o f  new cases in a defined population within a specified period o f  
time, usually one (1) year.
16. Prevalence
The proportion o f  a given population showing patent dracunculiasis infection at a given point 
in time. Prevalence data may be o f  little use with regard to dracunculiasis because the period 
o f patency is usually short and seasonal. Therefore, information collected during low 
transmission periods can be misleading. Prevalence should be measured at the period o f  peak 
patency.
17. Control
Reduction o f disease incidence in a defined area over a period o f  24 months through planned 
activities.
18. Elimination
Complete absence o f  new indigenous cases o f  patent dracunculiasis infection in a previously 
defined endemic area for a period o f  at least 24 months given the presence o f  an active 
surveillance system, including at least two annual village-to-village checks.
19. Eradication
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Global elimination o f  human dracunculiasis infection.
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Chapter 2 
2.0 LITERATURE REVIEW
2.1 HISTORY AND IMPORTANT DATES OF DRACUNCULIASIS
2.1.1 History
Dracunculiasis is an ancient disease quoted by many classical authors and mentioned in the 
Old Testament (Numbers Chapter 21). This nematode was known as a parasite o f  humans 
about 1530 B. C. The aduceus, which is the symbol o f  a physician is the staff o f  Hermes and 
contains coiled serpents on a staff. The coiled serpents are believed to represent the Guinea 
Worm (Muller, 1971).
During the 1980s, the World Health Organization gave its backing to United Nations 
coordination mechanisms and, in collaboration with its Member States, drew up a list o f  
attainable goals for the International Water Supply and Sanitation Decade. One o f  these goals 
was the eradication o f  dracunculiasis. This objective has been partially achieved. It was 
recently estimated that the prevalence o f  dracunculiasis has dropped from 1 0 -1 5  million 
cases per year at the beginning o f the 1980s to the present low figure o f  1 million cases.
The Member States o f  the World Health Organization then requested that every effort be 
made to interrupt transmission o f dracunculiasis before 1995. Once transm ission ceases, 
there will be a three-year monitoring period. It during this time no further cases are detected.
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WHO will issue an eradication certificate. For the last few years there has been a world 
eradication campaign (Abdou, 1982; Hopkins and Ruiz-Tiben, 1990).
2.1.2 Important Dates
Ever a subject o f  curiosity because o f  its apparently supernatural aspects, dracunculiasis has 
been documented since early history. In the 15th century BC, the first known mention o f  the 
disease is found in the AT urin Papyrus" which refers to the ancient Egyptian myth o f  the sun 
god Ra. A recent pathological examination o f  an Egyptian mummy clearly identified a 
calcified worm as Dracunculus medinensis. In the 14th century BC, the closing verse, o f  
three stanzas o f  a poem in the Sanskrit book Rig-Veda, attributed to Vasistha, allude to the 
guinea worm. Also, in the 11th century AD, Abou Ali ibn Sina (known in the West as 
Avicena) gives detailed descriptions o f  the disease, its treatment, its evolution and the 
complications caused by the worm being ruptured during extraction. Dracunculiasis 
occurred frequently in Persia during this period (htt:p//aepo-xdv- 
www.epo.cdc.gov/wonder/PrevGuid/mOOO 1960/m0001960.htm : 1 /18/00).
From the middle Ages through to the 18th century there were many varying opinions as to 
the nature o f  the "fiery serpents" - believed to be anything from exposed nerves to dead 
tissue, it was the celebrated Swedish naturalist, Carolus Linnaeus who first suggested that 
they were in fact worms. In 1870, Alexei P. Fedchenko became aware o f  the life cycle o f  D. 
medinensis and identified the copepod as its intermediate host. By the end o f  the 19th
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century, scientists had become well aware o f  how the disease was transmitted and had started 
to advocate suitable protective measures. Between 1926 and 1931, dracunculiasis was totally 
eradicated from Uzbekistan following a series o f  effective health education, water 
purification and carrier control programmes in Boukhara and the surrounding areas. No 
recurrence o f the disease has been recorded in this region since 1932. In the 1970s, 
dracunculiasis was eradicated in Iran. In 1984 dracunculiasis was eradicated in the Indian 
State o f Tamil Nadu, by 1989 in Gujarat, and by 1991 in Maharashtra (htt:p//aepo-xdv- 
www.epo.cdc.gov/wonder/PrevGuid/m0001960/m0001960.htm : 1/18/00).
2.2 EPIDEMIOLOGY AND TRANSMISION
This parasitic infection occurs most frequently in West Africa and in Western and Southern 
India, principally in the rural areas. Depending on the climate, dracunculiasis occurs in one 
o f at least three seasonal patterns. In semi-arid areas, transmission occurs in the rainy season. 
Since the incubation period averages about 12 months, the transm ission season remains 
synchronized with the annual period when the local environment is most receptive to the 
parasite. In areas where there are surface sources o f  water year-round, transmission usually 
occurs during the dry season, when the surface sources are scanty and most polluted. In some 
other areas, transmission may occur all year-round with very little seasonal variations. 
Because o f its life cycle and the year-long incubation period, the transm ission and clinical 
manifestations o f dracunculiasis are highly seasonal.
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Although the disease is easily diagnosed, and thus recognition o f  the problem is relatively 
simple, surveillance o f  dracunculiasis, paradoxically, is exceptionally poor. The lack o f 
ongoing case information is due in part to the remote rural nature o f  affected populations, 
their limited attendance at government clinics, and in some countries, the low government 
priority accorded to dracunculiasis. This situation could continue since the absence o f  a 
specific curative or preventive drug or vaccine removes a treatment incentive for villagers to 
visit health centers where their infection might be diagnosed and officially reported. In most 
endemic zones, less than 5 percent o f  cases are reported. M illions o f  cases are thought to 
occur annually worldwide, but systematic epidemiologic surveillance is needed to produce a 
reliable estimate o f  the annual global incidence.
2.3 ETIOLOGY AND LIFE CYCLE OF DR.4CUNCULUS MEDINENSIS
Although the discovery o f  the life cycle o f  Dracunculus medinensis has usually been 
credited to Fedchenko (1869), Manson-Bahr (1966) made the following statement: 
"Fedchenko (1869) is credited with the discovery o f  the transmission o f  the guinea worm, but 
probably, Manson was the original observer (1895). He believes that the stages figured by 
the former are those o f  Cucullanus spp (a parasite o f  fish), and not D. medinensis" (Hughes, 
1967). Hughes however agrees that Fedchenko correctly postulated that human infection is 
caused by the ingestion o f  infected copepods in drinking water. This was obviously because 
he was already aware that the life-cycle o f  some parasitic helminths involved alternative 
hosts, and he was looking out for such a host for the Guinea worm.
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Conversely, an investigator 75 years earlier did not know about alternative hosts, and so he 
(Manson) missed the complete story as unfolded by Fedchenko. Colin Chisholm (1795) also 
noted the occurrence o f  the "dracunculus or Guinea worm" and wrote: "The cause o f  this 
singular disease seems to be confined to the water o f  some wells". He described how filling 
up these wells prevented the spread o f  the disease, and continued; "In the water which 
contains the embryos o f  the dracunculi, the naked eye distinguishes innumerable animalculi, 
darting in every direction with astonishing force and rapidity; these on being subjected to 
examination with a small microscope, exhibit a very extraordinary figure, differing from any 
animalcule hitherto described". These were surely copepods. Thus, it was Fedchenko's better 
knowledge o f  parasitology and familiarity with the appearance o f  unparasitised copepods 
which enabled him to make the observations that had eluded Chisholm; that the Guinea 
worm embryos were inside copepods (Hughes, 1967).
Sir James Emerson Tennent gives another account o f  the mode o f  transm ission o f  Guinea 
worm. As the Colonial Secretary o f  the British Government in Ceylon from 1845 to 1850, 
Sir James Tennent noted in the course o f  a description o f  "parasitic worms" found there, that 
"of these entozoa, the Filaria medinensis or Guinea worm which burrows in the cellular 
tissue under the skin, is well known in the north o f  the Island, but rarely found in the damper 
districts o f  the south and west. The natives o f  these areas attribute the occurrence to drinking 
the waters o f  particular wells". Sir James Tennent thus records the fact that the mode o f 
infection/transmission seems to have been known on the African coast in at least the 
sixteenth century, and in Ceylon for some time before the establishment o f  British rule in the
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eighteenth century, thereby giving additional support to Dr. Muller’s contention that "the 
belief that transmission is associated with wells and water holes has been held since ancient 
times".
Sir James Tennent also goes further to suggest that "these pests in all probability received 
their popular name o f  Guinea worm from the narrative o f  Bruno or Braun, a citizen or 
surgeon o f  Balse, who about the year 1611 made several voyages to that part o f  the African 
Coast, and on his return, published, among other things, an account o f  the local diseases" 
(Gooneratne, 1969).
Bizarre as it appears, the life cycle o f  the Guinea worm, Dracunculus medinensis is actually 
very well adapted for the transmission o f  a parasite that utilises an aquatic intermediate host, 
which occurs principally in arid or semi-arid environments. Human infection occurs when 
copepods containing the infective stage larvae (L3) in water are ingested. The copepods are 
killed by gastric ju ices in the stomach and the larvae are activated and liberated and quickly 
pass through into the duodenum o f the human host within 4 hours after ingestion (Muller, 
1982).
They proceed to the abdominal and thoracic cavities where they begin maturing in 
connective tissue. Male and female worms mate about 3 months after ingestion. The male 
worms are believed to die at 6 months o f  age and then become encysted, calcified, or are 
absorbed. However, Brandt et al.,( 1990) demonstrated that male worms might survive for up 
to330
days in experimentally infected ferrets. The adult female worm, which measures about 70 cm 
long by 2 mm, lives in the connective tissues. At about 8 months female worms usually 
move down to the lower limbs, where the uterus containing first-stage larvae develops to fill
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nearly the entire adult worm.
21
In experimentally infected animals, the larvae are seen to penetrate through the duodenal 
wall in 10 to 13 hours post ingestion, and migrate via the mesenteries to the abdominal and 
thoracic muscles by about the fifteenth day (Rab, 1989). The larvae do not grow during this 
period, but there is probably a moult between the fifteenth and the twenty-first days. As they 
develop further, they begin to migrate towards the connective tissues o f  the axillary and the 
inguinal regions where they mature into adult worms. The size o f  the worms at this stage 
remains small and mating occurs between 80 and 100 days after infection (Muller, 1982). 
The males, (size: 1 to 4cm long) move into the deeper tissues. Within eight months after 
infection, the gravid female is filled up with developing eggs and with first-stage larvae by 
the tenth month. At this time, the worm begins its migration usually towards the extremities 
and is ready to emerge from the body between 10 to 14 months after infection. Before, the 
14th month, the gut becomes flattened and non-functional, and the whole worm is filled by 
the larvae-containing uterus (Muller, 1982).
When the worm (female) is ready to emerge, the anterior end o f  the worm  provokes the 
formation o f  a painful burning blister in the human skin. The worm emerges when this blister 
ruptures (especially upon coming into contact with water). Infected people frequently try to 
relieve the burning sensation by immersing the affected part in water. Contact w ith water 
causes the worm's uterus to rupture and stimulates the worm to expel larvae into the water. 
The process is repeated intermittently over several weeks.
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Numerous first-stage larvae (L,) are expelled into the water in a milky white stream. 
Estimates o f  the number o f  larvae contained in the uterus o f  a single worm range from 1.4 to
3.0 million. Not all the larvae are released at once, and it has been shown that about half a 
million larvae are released on first immersion in water. Out o f  water, the anterior end o f  the 
worm then becomes flaccid and dries up. When the affected part comes into contact with 
water again more larvae are expelled through the broken end o f  the worm. The number o f  
larvae expelled at each immersion decreases, and the worm is completely expelled within 
one to three weeks. This intermittent discharge and drying up o f  the blister aperture is an 
adaptation to increase the chances o f  some o f  the larvae finding copepods in the water. This 
process is one o f  the neatest adaptation in behaviour in all o f  the realm o f  biology, enabling a 
blind unmeditative burrowing worm to give her aquatic copepod-inhabiting offspring a  fair 
chance in life, even in deserts (Rab, 1989; Muller, 1971).
The first stage larvae, (640 by 13|i) remain active in the pond for about one week. Following 
ingestion by a Cyclops, the larvae penetrate the gut wall o f  the copepod and reach its 
haemocoel within one to six hours. They moult tw ice inside the copepods and reach the 
infective third-stage (L3) in 14 days. It is when man drinks this copepod-contaminated water 
that the cycle is repeated (Figure 1), (Rab, 1989; Muller, 1971; Brieger and Rosenweig, 
1988).
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The rate o f  development o f  the larvae in the copepod has been found to be temperature 
dependent. Temperatures above 24 °C and below 19 °C inhibit the growth o f  the larvae, 
which are then incapable o f  reaching the infective stage. It has also been observed that those 
larvae-containing copepods are sluggish in their movements and tend to sink to the bottom o f 
the ponds, as compared to the non-infected ones. There is also some evidence to suggest that 
the life span o f  the infected copepod is shortened (Rab, 1989).
2.4 CLINICAL MANIFESTATIONS AND PATHOLOGY
In most patients the first physical sign is the local lesion, accompanied by an intense burning 
pain usually relieved by immersion o f the affected limb in water. The blister fluid is 
bacteriologically sterile and contains numerous white cells and larvae. In uncomplicated 
cases, the worm emerges from the subsequent ulcer over a few weeks and then the lesion 
rapidly heals. I f  there is only one worm present, patency will last for only 4-6 weeks.
Unfortunately, secondary infection along the track o f  the worm in the tissues is very 
common, often with spreading cellulitis, and approximately 40% o f patients will be totally 
incapacitated for an average o f  6 weeks. More serious and permanent damage can follow the 
bursting o f  a worm in the tissues or as the result o f  bacterial infection. Dracunculiasis is 
unusual among infectious diseases in that the parasite does not appear to stimulate a pro­
tective response. Thus, that the same individual can be re-infected year after year.
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2.5.1 Clinical and Parasitological Diagnosis
Infected people exhibit no signs or symptoms until the female worm matures and is ready to 
emerge. The first manifestation o f  dracunculiasis is localized swelling at the spot where the 
mature worm will emerge. In over 90 percent o f  cases, it emerges somewhere on the legs or 
feet, although worms may emerge from any part o f  the body. Intense burning or itching 
accompanies the swelling, which develops into a blister within 1 or 2 days. This blister 
ruptures several days later and becomes a superficial ulcer. Infected people often immerse the 
lesion in water in an effort to relieve discomfort. The worm's uterus expels larvae when the 
affected part is exposed to water, a  process that may continue for several days to 3 weeks. 
Occasionally worms die before reaching the skin's surface and are absorbed, form aseptic 
abscesses, or become calcified, leaving cord-like masses. Generalized nonspecific symptoms 
may accompany the appearance o f  Dracunculus at the skin, but they are usually not severe. 
Such symptoms may include diarrhea, vomiting, skin rashes, or asthma.
The tissues near the blister become swollen, red, and very tender, probably as part o f  a 
largely allergic reaction. There is usually a secondary infection, which commonly spreads 
from the initial skin lesion to deeper tissues and may be accompanied by severe or fatal 
septicemia. Infected ankle and knee join ts can become contracted, leading to permanent 
crippling. Even in cases uncomplicated by secondary infection o f  the ulcer, the affected 
person may find it very difficult to walk and thus must give up his usual labors. On average, 
about 4-6 weeks may elapse before an uncomplicated infection heals completely.
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2.5 DIAGNOSIS
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Less frequent are other severe conditions or death resulting from Dracunculus infection. 
These conditions include septic arthritis, tetanus, gangrene, pulmonary scarring, and 
ophthalmic disease. A person may be infected by several guinea worms at the same time. 
Although each infection lasts a year, no effective immunity develops, and people at risk may 
be repeatedly infected year after year. Because o f  its unusual manifestation, guinea worm 
disease is easily diagnosed once the worm is ready to emerge.
Patients in an endemic area usually have no doubt o f  the diagnosis, as soon as. or even 
before, the first signs appear. Local itching, urticaria, and a burning pain at the site o f  a 
small blister are usually the first signs o f  infection. The blister bursts in about 4 days and 
active larvae, obtained by placing cold water on the resulting small ulcer, can be recognized 
under a low-powered microscope.
2.5.2 Immunological diagnosis
Diagnostic tests to detect the presence o f  Dracunculus at earlier stages have not been 
developed for routine use. However, laboratory investigators have reported positive 
fluorescent antibody tests 6 months or more before emergence o f  the worm (Belcher. 1982). 
Another nonspecific aid to diagnosis may be eosinophilia o f  10-15 percent. In general, 
however, very little biomedical research on dracunculiasis has been carried out. thus making 
interpretation o f  findings reported in the literature quite difficult. Immunological methods are 
not useful in practice. ELISA and SDS PAGE/Western blotting worked well in one trial for
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patent infections (Bloch et al., 1983) and the fluorescent antibody test using deep frozen first 
stage larvae diagnosed prepatent infections in monkeys (Muller, 1971).
2.6 TREATMENT
2.6.1 Surgery
Guinea worms have been wound out on sticks since antiquity (e.g. in the Rig Veda o f  about 
1350 BC). Provided that bacterial infection or other complications have not occurred, 
regular winding out o f  the worm on a small stick, combined with sterile dressing and 
acriflavine cream, usually results in complete expulsion in about 4 weeks with little loss o f  
mobility. T reatment should be commenced as soon after emergence as possible (Magnussen, 
et al., 1997). Sometimes worms can be seen and surgically removed before emergence while 
there is no tissue reaction against them.
2.6.2 Chemotherapy
There is no evidence that any chemotherapeutic agent has a direct action against guinea 
worms. No drugs have proved effective in killing the adult worm prior to emergence, 
although some have shown experimental promise in reducing inflammation and facilitating 
extraction o f  the worm. Eberhard, et al., (1989) came to the conclusion that it appears 
existing drugs commonly used to treat helminthic infections are poor candidates for use in 
the campaign to eradicate guinea worm disease. This was after they evaluated the potentials 
o f 4 chemoprophy lactic drugs for use in the treatment o f  dracunculiasis by the Dracunculus 
insignis-ferret model.
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27
Niridazole, metronidazole, thiabendazole, levamisole, bitoscanate, and mebendazole have 
been tested in humans within the last 7 years. O f these compounds, only thiabendazole if 
given as a short (2-day) course o f  treatment, would make patient compliance more likely. 
One study reported that mebendazole (7-day course) resulted in significant symptomatic 
improvement. The action o f  niridazole appears to be largely one o f  reducing inflammation. 
For the most part, however, these drugs are expensive and may not be available locally. 
Large-scale controlled clinical trials have not been conducted, and the results o f  the smaller 
studies are difficult to interpret because o f  different patient selection methods, wide variation 
in criteria o f  efficacy, lack o f  control groups, and high drop-out rates.
The majority o f  infected people neither seeks nor receives medical care from qualified 
physicians or nurses. Patients often consult healers or resort to the traditional technique o f  
extracting the worm by rolling it a few  centimeters each day around a stick or gauze. 
Infection often results if  the worm breaks. Some physicians prescribe chemotherapy and 
make multiple incisions under local anesthesia to extract the emerging worm. Antibiotics 
may be given to treat secondary bacterial infections, and tetanus prophylaxis is strongly 
advocated.
Although there are no drugs to treat or prevent dracunculiasis, transm ission is easily 
interrupted by simple measures such as behavioural changes, protecting wells, tanks and 
water sources, filtering water before drinking it, or (when possible) treating water with
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28
“Temephos" (a biodegradable organophosphorous compound) to kill the copepods. These 
have made it possible to eliminate the disease from many affected areas and to aim at total 
eradication.
However, many compounds, including thiabendazole, niridazole, metronidazole, 
mebendazole and albendazole, have been reported as hastening the expulsion o f  worms and 
may act as anti-inflammatory agents. Ivermectin had no action against pre-emergent worms 
(Eberhard, et al., (1989).
2.7 SOCIAL AND ECONOMIC EFFECTS
Cases o f Guinea worm disease have been declining in much o f  Africa since the inception o f  
the Guinea worm Eradication Programs in 1983. Unfortunately, the infection still remains a 
serious public health problem in some countries. According to the US Centres for Disease 
Control and Prevention (CDC), Guinea worm is especially problematic in southern Sudan, 
where civil war has impeded efforts to eradicate it. There were 66,097 reported cases o f  the 
disease in Sudan in 1999. Outside o f  Sudan, there were 12,097 cases o f  Guinea worm during 
the first six months o f  2000, 18 percent less than the 14.828 cases reported during the same 
period in 1999. However, there was a slight increase in the number o f  cases in Ghana during 
the first six months o f  2000 (Ruiz-Tiben, August 2000 -  Personal communication).
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Guinea worm has been an officially reportable communicable disease in Ghana since 1960 
(Diamensu and Nyaku, 1998). The Ghanaian government launched a Guinea worm 
Eradication Program in 1987. Despite the political will and massive international support, 
there is unfortunately an apparent lapse in the control areas. This allowed for resurgence o f  
the disease in some communities. For 1997, the nation recorded 6,844 cases o f  dracunculiasis 
during the period January-August from 703 villages, an increase o f  73% over the same 
period in 1996. The region most afflicted, the Northern Region, reported a total o f  5,977 
cases from 534 villages, representing an increase o f  63.8% over 1996 when 3,902 cases were 
reported from 455 villages. These dramatic increases in reported cases o f  Guinea worm in the 
country called for the intensification o f  efforts for the disease eradication.
2.7.1 Cultural and Traditional Beliefs
In village life, an enigma truly remains as to why some individuals are Guinea worm-free, 
while the majority are annually afflicted, and even why some unfortunate few  are so 
massively infested. Following the UNO Decade o f  Water, the question may be moot, but it 
does recognize fertile ground for the growth o f  cultural belief, or folk rationale, to "explain” 
dracunculiasis and place it in a belief system. In Ghana, women in endemic villages, 
although zealously indoctrinated by visiting health education officers, cannot easily accept 
that invisible objects (copepods) in drinking water cause the disease. Ostensibly, they 
acquiesce to the superposed dicta o f  the educator, but to them, a cultural matrix o f  “jealous 
rivals”, “enemies”, and “witches” is an equally plausible causation (Hunter. 1996).
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A study in I mo State, Nigeria, showed that 35% o f  the disease victims implicated their 
enemies whilst 37% o f  them believed that the disease was an inherited family trait. 
Ironically, only ll% accep ted  the polluted water explanation (Nwoke, 1992). Local people 
often believed that the worm is a natural phenomenon, a “natural part o f  the body like a 
tendon" an innate part o f  the human anatomy (Adeniyi, et al., 1992). This idea matches those 
o f the early Persian, Arabic, and Greco-Roman physicians who reported that dracunculiasis 
was anatomically o f  nerve-like origin, resembling varices (veins) or a “piece o f  corrupted 
nerve”.
Bierlich (1995), carried out an anthropological study o f  beliefs and practices concerning 
infection with Guinea worm (Nyarfu/Nierifu), in two rural Dagomba communities in the 
Northern Region o f  Ghana. He found that the local people do not attribute Guinea worm to 
water. The general understanding is that Guinea worm is an innate part o f  human anatom}'. It 
is not seen as an alien presence in the body. Guinea worm is rather said to be 'in people's 
blood’, and sooner or later to 'stand up'. Guinea worm is considered an 'inevitable' feature o f 
living ('Guinea worm is in the human blood'). These observations are very different from 
those o f  biomedicine, which hold that 'Guinea worm is a disease’. Omar et al.. (1993) 
examined the knowledge, attitudes, practices, and beliefs o f  the people concerning Guinea 
worm disease and its transmission, prevention, and treatment. They found that most people 
could describe the cause and treatment. More than 58% thought dirty or bad water was the 
main cause, while up to 63.2% believed that contaminated drinking water was the cause
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31
Few people clearly understood how Guinea worm is transmitted. People received treatment 
from the nearest health unit (42%), traditional healers (34%), self-medication (16%), and 
drug shops (8%).
2.7.2 Socio-Economic Aspects o f Dracunculiasis
The basic nature o f  Guinea worm disease in Ghana shows its long-overlooked serious 
clinical aspects, and the many environmental and social influences that explain its 
persistence in the face o f  control efforts. It is a neglected disease o f  remote rural areas. The 
socio-economic impact o f  the malady has, however, assumed a more quantifiable aspect in 
recent times. Although the disease is rarely fatal, and less than 1% o f  victims suffer 
permanent disability, it prevents large numbers o f  people from farming or attending school. 
It is a severely disabling parasitic disease, responsible for heavy economic losses and serious 
social repercussions in endemic areas (Ogunniyi and Amole, 1990; Chippaux et al., 1992). 
The skin lesions and subsequent scars produced by the emerging mature female worm alone 
could be both a social stigma and embarrassment. Infected people are often incapacitated for 
several weeks by secondary infections associated with the emergence o f  the worm.
2.8 CONTROL MEASURES
Dracunculiasis transmission is considered to be "water-based”. The cycle o f  transm ission 
requires that; (1) infected individuals immerse the mature emerging female worm in water 
used for drinking, (2) suitable copepod species are present in that water source under optimal
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conditions, and (3) someone drinks water containing copepods infected with mature 
Dracunculus larvae. Any break in this chain o f  events will interrupt transm ission o f  
dracunculiasis.
Effective personal protective or prophylactic measures that may be used include boiling the 
drinking water or straining 'it through a cloth to remove copepods. The utility o f  these 
measures for a large-scale attack on the problem is somewhat diminished by their 
dependence on intensive educational efforts and by their inconvenience or cost (e.g.. farmers 
quenching thirst in fields, firewood needed for boiling water). Health education, including 
community organization, might also be employed to encourage residents o f  affected villages 
to prevent people suffering from the disease from entering, and thereby contaminating, 
sources o f  drinking water.
Effective control measures include the periodic chemical treatment o f  water used for 
drinking in affected villages to kill copepods. Temephos (Abate) is the insecticide most 
commonly used for this purpose. At concentrations o f  1 part per million in stagnant surface 
sources o f drinking water, temephos kills copepods; is harmless to vegetation and fish; and is 
tasteless, colorless, and odorless in drinking water; and has a wide margin o f  safety for 
ingestion by humans. Moreover, the compound has been used extensively in West Africa to 
control the blackfly vector o f  onchocerciasis.
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Thus far, however, the most effective means o f preventing dracunculiasis has been to provide 
safe water supplies. Such protected water sources prevent contamination o f  the drinking 
water by larvae from infected people, thereby breaking the chain o f  transmission. Provision 
o f  safe water via piped sources and protected bore and tube wells, along with the destruction 
or conversion o f  contaminated step wells, successfully elim inated dracunculiasis from large 
areas in the southern part o f  the Soviet Union in the 1920s and 1930s. In Nigeria, 
construction o f  piped water for a town o f  30,000 people in the 1960s reduced the incidence 
o f  dracunculiasis from over 60 percent to zero within 2 years. In several other instances, 
dracunculiasis has been eliminated or drastically reduced as a side benefit o f  efforts to bring 
safe drinking water to rural populations who happen to suffer from the disease.
The current International Drinking Water Supply and Sanitation Decade offers a unique 
opportunity for an attack on dracunculiasis. In the context o f  the Decade, the provision o f  
safe drinking water is intended for all by 1990. Hence, there is no need to justify providing 
safe drinking water solely as a means o f  eliminating dracunculiasis, only to encourage 
endemic countries to consider this disease when assigning relative priorities to areas where 
elimination o f  the disease would occur in addition to other benefits. Indeed, the number o f  
villages in which dracunculiasis is endemic is estimated to be less than 10 percent o f  all 
villages targeted to receive safe drinking water during the Decade.
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2.9 THE PARASITE
2.9.1 Morphology
Nematodes are ubiquitous, unsegmented, pseudocoelomate worms, and probably the most 
abundant multicellular animals living today. Among these are some o f  the major parasites o f 
vertebrates. There is however a difficulty in describing or recognising nematodes species. 
This is generally due to their small size and in the extreme uniformity o f  both internal and 
external anatomy as well as morphology. Often, species determinants must be based on 
biochemical attributes or morphological details not readily visible, such as the size and 
placement o f  microscopic sensory structures or morphological details o f  the excretory 
system (Pechnenik, 1991).
Generally, the body organization o f  the Nematoda is sim ilar throughout the Phylum. All 
actively moving nematodes have cylindrical bodies that usually taper at the anterior and 
posterior ends. The constancy o f  the structure o f  the nematodes is not limited to the general 
body shape, for no matter what the size o f  the worm, the form and arrangement o f  the 
internal organs is sim ilar (Obeng, 1997).
The sexes are separate in the Dracunculus spp. The male is rarely seen. Records claim it to 
be about 40mm long and only 0.4mm in width. The female is the better known o f the two. As 
an adult, it is one o f  the longest human nematode parasite, with an average length o f  about 
60cm and a width o f  1.5 - 1.7mm. The head end of the worm has glands and a thick 
protective cap (Obeng, 1997). According to Muller (1971) and Hunter (1996), the mature
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female o f  the dracunculoid nematode, D. medinensis, measures 500-800 mm by 1.0-2.0 mm 
(Figure 2). The anterior terminal mouth has a triangular oval opening surrounded by a 
quadrangular cuticularized plate, and an internal circle o f  four (4) double papillae. The 
digestive system is simple with a bulbous oesophagus, long simple intestine and an anus. The 
worm feeds on fluids and juices in the body o f  the host. There is a simple nervous system, 
but no easily identifiable excretory system. The principal parts o f  the female reproductive 
system consist o f  two ovaries, each leading into an oviduct and a uterus that merge to form a 
vagina that is attached to an ovijector attached to the body wall at the vulva. It is suggested 
that females often reproduce without mating (Obeng, 1997), but this is well recognized to be 
incorrect (Mark L. Eberhard, 2001 -  personal communication).
The vulva opens halfway down the body but is non-functional in the mature worm. The 
uterus has an anterior and a posterior branch; it is filled with 1 -3 million embryos and occu­
pies the entire body cavity (pseudocoel), the gut being completely flattened (Muller. 1971). 
The worm is pale white, thread-like and soft (Flunter, 1996). Males recovered from 
experimental infections in animals measure 15-40 x 0.4 mm. The tail has 4(36) pairs o f  pre- 
anal and 4-6 pairs o f  post-anal papillae; the sub-equal spicules are 490-750|im  long with a 
gubernaculum measuring about 117|im.
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The phylogenetic systematics o f  D. medinensis has seen some form o f metamorphosis, as has 
been the case for most organisms. In literature therefore, the Nematoda is sometimes listed as 
a class within another phylum, the Aschelminthes. The Aschelminthes are often referred to as 
the "Cavity worms" because they posses a pseudocoel. The majority o f  groups contained 
within the Aschelminthes (Nematoda, Rotifera, Gastrotricha, Kinorhyncha, Nematomorpha, 
Acanthocephala and Gnathostomulida) are now generally considered more distantly related, 
so that each class has been elevated to the phylum status (Pechenik, 1991). Thus, D. 
medinensis has been assigned the following taxonomic groups.
36
2.9.2 Taxonomy
PHYLUM: Nematoda
CLASS: Secementea.
SUBCLASS: Spiruria.
ORDER: Camallanida.
FAMILY: Dracunculidae.
GENUS: Dracunculus.
SPECIES: medinensis.
SCIENTIFIC NAME: Dracunculus medinensis.
COMMON NAME: Guinea worm.
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37
The transmission o f  D. medinensis requires the involvement o f  a water flea as the 
intermediate host. This flea is a minute crustacean, the size o f  a pinhead, pear-shaped, a free- 
living fresh-water cyclopoid copepod, with different genera and species (Figure 2). 
Dracunculus medinensis however exhibits a high level o f  host specificity and only a few 
species act as vectors in nature. Cyclopoid copepods (Cyclopoida) are known as the 
intermediate hosts o f  more than ten genera o f  the Cestoda and Nematoda. Two among these 
are important parasites o f  man; Diphyllobothrium latum (the broad fish tapeworm) and 
Dracunculus medinensis.
Mesocyclops leukarti (Claus) is widely reported as the common intermediate host in India 
and Africa (Onabamiro, 1951; Muller, 1970). O ther reported hosts include species o f  the 
genera Mesocyclops Sars, Thermocyclops Kiefer (Onabamiro, 1951) and Metacyclops Kiefer 
(Steib, and Mayer 1988). The correct identification o f  the copepod intermediate hosts is 
therefore not only important in mapping out the geographical distribution and spread o f  the 
disease, but also vital in the development o f  eradication programmes that aim to combat the 
disease by vector control (Boxshall and Braide, 1991).
Copepods (classified as Macrozooplankton, Sensu stricto) are subdivided into Calanoids, 
Cyclopoids and Harpacticoids. O f these groups, only cyclopoid copepods serves as 
intermediate hosts o f  D. medinensis. Recent progress in copepod systematics has refined the
2.10 THE VECTORS OF DRACUNCULUS MEDINENSIS
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level o f  taxonomic resolution o f  these freshwater copepods and it is now known that 
Mesocyclops leukarti does not occur in either Africa or India (Keifer, 1981; Van de Velde, 
1984). There is, therefore, an obvious need to record these taxonomic changes, to review 
earlier records, and to update the nomenclature o f  the hosts where possible (Boxshall and 
Braide, 1991).
The various groups have been observed to have diverse feeding regimes, often modified in 
the course o f  development. Cyclopoids have an erratic jumping motion which makes them 
more conspicuous than the gliding motion o f  calanoids (McCullough, 1982). The broad 
taxonomic grouping o f  copepods is as follows.
38
Phylum: Arthropoda
Subphylum: Crustacea
Class: Copepoda
Order: Cyclopoida
Family: Cyclopidae
Thus, cyclops belongs to the Cyclopoid copepods, one o f  the orders in the class Copepoda. 
The Copepoda is included in the subphylum Crustacea that also include such familiar 
invertebrates as crabs, shrimps and lobsters (Barnes, 1968). Freshwater Cyclopoid copepods 
are minute, pinhead-sized crustaceans which are biologically very successful, comprising
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many genera with diverse feeding habits. They are found world wide and almost exclusively 
in standing or slow-flowing, marine, brackish and freshwater bodies. The free-living 
cyclopoid copepods (often referred to as cyclops), have pear-shaped bodies, comprising a 
cephalothorax, an abdomen and a telson with a tail which has two caudal rami. The sexes are 
separate and the eggs hatch into typical nauplius larvae, which are then succeeded by several 
metanauplius stages before moulting into the first o f  five successive copepodite stages 
(McCullough, 1982).
Muller (1971), listed 17 species o f  copepods that potentially act as intermediate hosts in 
different Dracunculiasis endemic areas. More recently Steib, (as cited in Olsen, 1993). 
working in Burkina Faso, added two more species (Thermocyclops incisus and Metacyclops 
exsulis) to the list o f  potential vectors. In Africa however, approximately 150 different 
species o f freshwater cyclopoids have been described. O f these only 60 are prevalent in areas 
endemic for Guinea worm disease. Probably, only 34 o f  these species are frequent vectors. 
Generally, only members o f  the genera Thermocyclops and Mesocyclops are regarded as 
truly planktonic forms and can therefore act as the most important vectors o f  the disease 
(Kiefer, 1978; as cited in Oslen, 1993).
McCullough (1982), states that only large predatory species can readily ingest Dracunculus 
medinensis larvae and can therefore act as potential intermediate hosts. O f these carnivorous 
species, the older and larger copepodid stages are more predatory than the younger smaller
39
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ones. Also, that in each endemic zone usually one o f  the local predator species is the 
dominant intermediate host by virtue o f  its preferred habitat and seasonal population 
dynamics or both.
Furthermore, Boxshall and Braide (1991), working on the freshwater Cyclopoid copepods o f 
Nigeria, identified forty valid and four non-valid vector species o f  dracunculiasis. Species in 
the genera Thermocyclops, Mesocyclops and Metacyclops are often implicated as 
intermediate hosts o f  D. medinensis. Boxshall and his coworkers were however quick to 
point out that the often implicated (and misidentified) host, Mesocyclops leukarti does not 
occur in either Africa or India. This view is also held by Olsen (1993), who states in his key 
"Vectors o f Guinea worm disease in tropical Africa - A key to the species o f  Thermocyclops 
and Mesocyclops", that o f  the 13 taxa o f  this genus described for Africa, most forms were 
recorded as Mesocyclops leukarti, which does not in fact occur in tropical Africa. Chippaux 
(1991: as cited in Boxshall and Braide, 1991), also identified 14 species o f  cyclopoids out o f 
which 4 were suitable intermediate hosts for D. medinensis. O f these, Thermocyclops 
oblongatus appeared to be the most common, especially in ponds, followed by T. neglectus 
whilst T.crassus consimilis was considered a minor intermediate host. T. emini also played 
an important role as an intermediate host, especially in rivers at the beginning o f  the dry 
season when the rivers had stopped flowing (Boxshall and Braide, 1991).
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41
Other species o f  copepods known to transmit Dracunculus medinensis are Thermocyclops 
nigerianus and T. hyalinus (Onabamiro, 1951). With respect to the food and feeding habits 
o f  copepods, the work o f  Klugh (1927), is believed to give a comprehensive review o f  the 
food o f  freshwater Entomostraca (Birge, 1897; as cited in Fryer, 1955).
Horse dung infusions (essentially protozoan cultures) appearto have been the chief source o f 
food used in cultures. Coker (1933; as cited in Fryer, 1955), remarks that this medium is 
satisfactory for the rearing o f  Acanthocyclops vernalis and Eucyclops (= serrulatus), but 
states, "we had reason to doubt its effectiveness with respect to the fertility o f  adults reared 
as to its general sustainability for A  viridis". Later Coker is said to have noted that he had 
satisfactory results by the addition o f  unicellular green algae and chopped fragments o f  the 
filamentous green alga Mougeutia spp. to the culture medium (Fryer, 1955). Fryer also 
revealed that individual species o f  copepods had preferred food material. The carnivorous 
species included Macrocyclops albdus, M. fuscus, Acanthocyclops viridis, A. vervalis, 
Cyclops strenuus and Mesocyclops leukarti. The herbivorous species were mostly from the 
genus Eucyclops and some Microcyclops.
The physico-chemical properties o f  copepod habitats depend on the general pattern o f 
seasonal events. As Burgis (1971), points out, in the temperate lakes, the seasonal changes 
are due mainly to the incident solar radiation, and the consequent changes in water 
temperature leads to an alternate building-up and breakdown o f  thermal stratification. In the
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tropical regions however, incident solar energy is high throughout the year, thus, diurnal 
stratification is o f  greater significance than seasonal changes. This phenomenon is 
pronounced in large bodies o f  water. In the small ponds that are often used as sources o f  
drinking water, the almost constant mixing o f  the water produces an environment remarkably 
homogenous, with stable physico-chemical conditions. The only apparent seasonal changes 
are those associated with increased inflow during the rainy season and the consequent 
increase in volume, often coupled with increased turbidity (Fryer, 1955).
2.11 ZOONOSIS
Female worms o f  the genus Dracunculus have been reported as emerging from  a wide range 
o f  mammals and reptiles from many parts o f  the world, both endemic and non-endemic for 
the human disease. Those found in reptiles clearly belong to other species but the situation 
in regard to those in mammals is not clear. For instance, Guinea worm is common in wild 
carnivores in North America and the species was named D. insignia by Leidy in 1858. and 
although distinct, there are few morphological features that separate this from the human 
species.
There have been two documented cases o f  clearly zoonotic infections, from Japan in 1986 
and from Korea in 1926. In both cases the patients had eaten raw freshwater fish, which 
have been proved experimentally to be capable o f  acting as paratenic hosts. In most highly
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endemic areas occasional infections in dogs and donkeys with what is presumably the human 
parasite have been reported but there is no evidence that they have any part in maintaining 
transmission. The parasite can still be found in dogs in the formerly endemic areas o f  Tamil 
Nadu in India and the central Asian republics o f  the former Soviet Union, but no new human 
cases have been reported, so it is not thought that this will be a problem once world 
eradication has been achieved.
2.12 GUINEA WORM ERADICATION PROGRAMMES (GWEPS)
2.12.1 Global Perspective.
Edingbola et al.,( 1988), estimated the worldwide incidence o f dracunculiasis at 5-15 million 
per year, all in the poor and remote rural area o f  Africa and the Indian subcontinent. The 
vagueness o f  this estimate is itself evidence o f  how little was correctly known about the 
prevalence and detailed distribution o f  the disease. However, the World Health Assembly 
Resolution o f  May 1986 (Res. No. 3G. 21) in support o f  the elim ination o f  Guinea worm, 
and the World Health Organization Water and Sanitation Decade, (which ended in 1990), 
have helped focus attention on the disease and the problems involved in its study (Smith et 
al., 1989).
In 1991, the Forty-fourth World Health Assembly declared the goal o f  eradicating 
dracunculiasis (guinea worm disease) by the end o f  1995 (Resolution WHA 44.5). In 1988
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the World Health Organization (WHO) Regional committee for Africa set itself the goal o f  
eradicating the disease in all 17 remaining endemic countries (except Sudan which is in the 
WHO Eastern Mediterranean Region) by 1995 (Resolution AFR/RC38/ R13). Apart from 
these 17 African countries, Indian and Pakistan were the other countries still with the disease, 
which is only transmitted to humans when they drink water from stagnant ponds, step wells 
or cisterns (Hopkins and Ruiz-Tiben, 1991).
Beginning in 1986, 1987 and 1988 the Global 2000 Project o f  the Carter Presidential Centre 
assisted dracunculiasis eradication programmes in Pakistan, Ghana and Nigeria respectively, 
in collaboration with the ministries o f  health o f  these countries. The WHO Collaborating 
Centre for Research, Training and Eradication o f  Dracunculiasis at the Centres for Disease 
Control and Prevention also provided extensive technical assistance to these programmes. 
Eradication o f  dracunculiasis is particularly feasible for the following reasons:
1. There is no human carrier state beyond the one-year incubation period.
2. Transmission could be seasonal, depending on the climate and topography o f  the area in 
question
3. Active detection o f  individuals with worms protruding from skin lesions is a sensitive means 
o f assessing the presence o f the disease in the endemic villages.
4. The methods for controlling transmission are simple and can be targeted effectively.
5. The total estimated cost o f  global eradication is moderate - about US$ 75 million (Hopkins 
and Ruiz-Tiben, 1991).
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Target dates for eliminating the disease were set at 1990 for Pakistan, 1993 for Ghana and 
1995 for Nigeria. In Pakistan 408 villages with a total o f  2 400 cases for 1987 alone was 
recorded, while 640 000 - 650 000 case from 6 000 villages were recorded from Nigeria. 
Ghana conducted a national search for cases in 1989/90, and found that almost 180 000 cases 
had occurred in the past year in about 6 500 villages. Each endemic country was mandated to 
decide on the most appropriate mix or combination o f  the available interventions. These 
included Health Education, Rural Water Supply, the use o f  Cloth Filters and Vector Control 
that it can afford or seek external help. No single intervention will work anywhere, and 
"problem" villages are to be dealt with as they become apparent.
With the eradication o f  the disease from India and Pakistan, the main focus o f  attention is 
now on Africa. In these countries, under the auspices o f  the WHO Regional Office for 
Africa, the respective ministries o f  health resolved in 1988 to eradicate dracunculiasis by the 
end o f  1995. This has since been rescheduled year after year, with the focus now on 
December 2002.
2.12.2 The Guinea Worm Eradication Programme in Ghana
The campaign to eradicate the GWD in Ghana was launched by the government in 1989. 
This policy is based on the W1IA Res. 39.21, which was adopted by the WHA in May 1986 
and endorsed by Ghana as a member state o f  the WHO. The original target date o f  December 
1993 was later shifted to December 1995. A myriad o f  problems including the 1994 ethnic
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conflict in the Northern Region and operational fatigue as well as funding problems 
prevented the achievement o f  the target, even though it is reported to have achieved a 95% 
reduction in 1996. Against this background, the President was obliged to visit Gushiegu, the 
most endemic town in the country in 1997 to re-launch the programme. Unfortunately, the 
Programme had to be re-launched again in April 1999 in the Afram Plains District o f  the 
Eastern Region due to the unusually high number o f  reported cases. The new  date for 
eradicating Dracunculiasis in the country was once again set at 31 st December 1999; it has 
since been shifted to December 2002.
In his opening address at the 1998 annual National Review Meeting o f  the GGWEP on 27th 
September 1998, HE Vice President Professor John A tta Mills declared: “The scourge o f  
Guinea worm has been a source o f  worry for Ghanaians, especially those who do not have 
clean water". He, however, acknowledged that the provision o f  good drinking water will not 
only eradicate the disease, but can also lead to a reduction in other water related diseases. 
The Vice President therefore called for a vigorous health education effort to eradicate the 
disease since the disease thrives in an ignorant and apathetic society.
The Plan o f  Action o f  the GWEP in Ghana states the methodology, strategy, targets and the 
estimated cost o f  achieving the objectives o f  total eradication o f  the disease by the year 1995. 
The programme in Ghana began identifying and training resident village volunteers (VVs) to 
report cases o f  Dracunculiasis in endemic communities in the early part o f  1989 (Hopkins
46
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47
and Ruiz-Tiben, 1991), control measures were directed towards freeing domestic water 
sources o f  copepods and preventing infected people from contaminating water supplies 
(Quashie, 1982).
The G WEP in Ghana is supported by the government as well as donor agencies such as the 
Carter Center in Atlanta Georgia, Bank for Credit and Commerce International (BCC1) and 
the Sasakawa Global 2,000 Project. Since the government recognized the need to eradicate 
dracunculiasis as a means o f  poverty removal/reduction, the National Guinea Worm 
Eradication Programme was incorporated into the M inistry o f  Health (MOH) with an 
autonomous adm inistration headed by a National G WEP Coordinator. The mandate o f  the 
programme was to evolve a focal control strategy for eradicating the disease by 31st 
December 1995.
The intervention strategies adopted were based on seven points:
1. Surveillance.
2. Health Education (HE).
3. Provision o f  safe drinking water
4. Training.
5. Monitoring and evaluation.
6. Research.
7. Vector control.
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48
The main emphasis as o f  July 1995 was all but research. An extraordinary level o f  public 
mobilization was achieved in June 1988 when the Head o f  State, Fit. Lt. Jerry John Rawlings 
spent eight day visiting 21 endemic villages in the highly infected Northern Region, 
promoting the goals o f  the national eradication campaign: an exceptional degree o f  
involvement by any Head o f  State in combating any disease (Report on 1DWSSD Impact on 
Dracunculiasis, October 20, 1989). Ghana also began distributing tens o f  thousands o f  copies 
o f a manual to Secondary schools in 1989 for teaching about the guinea worm disease. Both 
Ghana and Nigeria are emphasising on Health Education, Community Mobilization and 
Rural Water Supply in their intervention so far. An extensive use o f  temephos (Abate) in 
selected villages began in 1991.
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49
3.0 SOCIO CULTURAL STUDIES
3.1 MATERIALS AND METHODS
3.1.1 Study Locale
The study was carried out in 7 endemic villages in 4 districts, including Kukuo. Diare and 
Vognayili in West Dagomba, Gushiegu and Karaga in Gushiegu-Karaga, Savelugu in 
Savelugu-Nantong, and Nyankpala in the Tolon-Kumbumgu district in the Northern Region 
o f Ghana (Figure 3). These villages had population sizes o f 4,370; 9,514; 250; 7,511; 1,250; 
27,478; and 6,776 respectively (National Population Census, 2000). Lying approximately 8- 
10°N latitude in the Savanna woodland, the region receives 40-45 inches (1015-1142mm) o f 
rainfall annually. There is only one season o f  rain, mostly in April-October, associated with 
the air mass movements o f  the Inter-T ropical Convergence Zone (ITCZ). Data was collected 
over one month period (Mid-June to Mid-July 1999), at the tail end o f  the most recent 
transmission season. These districts were specifically chosen for their high incidence o f 
dracunculiasis. Complications caused by secondary infections were also common. These 
communities utilized water sources mainly typical o f  the traditional modes o f  water supply 
based on the collection and storage o f surface water in ponds, large contaminated earthen 
dams, or reservoirs. These water sources are the essential points o f  dracunculiasis 
transmission in the dry season.
Chapter 3
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50
3.1.2 Retrospective Studies for Disease Prevalence
Data collection involved interview and focus group discussions. A standardized 
questionnaire was developed and used to ascertain the peoples’ knowledge base with respect 
to perceptions on disease causation, prevention, treatment, and transm ission (Appendix A). 
Here, face-to-face interviews were held with respondents. Disease prevalence was estimated 
by assessing the Guinea worm status o f  one person interviewed per household. Mostly, it was 
the head o f  household who were taken as respondents. However, in the absence o f  the head, a 
reliable informant was interviewed. In order to compare responses o f  individuals who have 
or are currently afflicted with Guinea worm disease with those who have never contracted it, 
the households, and therefore, the respondents were randomly chosen. All the interviewers 
spoke both Dagbani (the local language/dialect), and English. A total o f  383 
/households/people, made up o f  226 males and 157 females living in the four districts were 
involved in the study.
A focus group discussion session was held in each village, with 8-15 adults (men and 
women) between 29 and 50 years o f  age, and 10 children, ages 11-15 years old. Information 
collected from respondents consisted mainly of: beliefs and practices, knowledge base 
concerning causes, mode o f  transmission, perceptions on curability and prevention, source o f 
respondents’ knowledge, source o f  water, and level o f dependency on it. Risk factors (human 
practices and beliefs), hypothesized to have some relationship with the disease causation, 
were selected and grouped into demographic characteristics. Some socio-economic factors
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51
examined included occupation and educational level o f respondents. The relative prevalence 
o f Guinea worm with respect to these factors and practices were then evaluated.
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3.2 RESULTS
52
3.2.1 Prevalence o f Guinea Worm Disease
Overall, 244 (64%), o f  the respondents reported to have had Guinea Worm Disease (GWD) 
at least once in the past two years, with 36% escaping infection. Out o f  this number, 43%  o f 
respondents had the disease in the current season (September 1998 - August 1999), with 24% 
and 32% having been afflicted in the previous year as well as both the previous and current 
year, respectively. With respect to gender, 226 (59%) o f  the victims were males, and 157 
(41%) were females.
3.2.2 Seasonal Distribution of Guinea Worm Disease
Cases o f dracunculiasis were recorded in 10 o f  the 12 months o f  the year. The highest 
number o f cases was recorded in May (25%), while no cases were recorded in August and 
September. Notably, the disease appears to begin in October (0.8%). and gradually increases 
every month thereafter, peaking in May o f  the next year before sharply dropping in 
magnitude and disappearing completely by the end o f  July. Most cases were observed from 
December to May (88%), whilst the period July to November recorded only 5% o f  all cases. 
Thus, there appears to be a very long period o f  disease transm ission, w ith a peak in April to 
May each year (Figure 4).
The negligible occurrence o f dracunculiasis in the study area within the period July to 
October (0.2%), with a complete absence in August and September is a notable feature. It is
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important to note that this is the peak o f  the rainy season in the region. Thus, most 
respondents might be harvesting and storing rainwater from their roofs for domestic use.
3.2.3 Occupational Distribution o f  Guinea Worm Disease
A majority o f  the Guinea Worm victims were farmers, with a considerable number being 
pupils, students, housewives, and traders. Thus, 94 (39%) were farmers, 41 (17%) pupils, 40 
(17%) housewives, and 30 (12%) traders (Figure 5).
3.2.4 Perceptions o f Disease Causation
To ascertain the knowledge base o f  the respondents regarding dracunculiasis, they were 
asked some pertinent questions such as; “ What do you think causes GWD? ”, "How is GWD 
spread?”, “ Can GWD be cured?’’, and “Can GWD be prevented?”. A majority o f  
respondents (62%) alluded to the fact that dracunculiasis could be acquired by drinking water 
from a pond/dam. This state o f  awareness could be due to the active status o f  the Guinea 
Worm Eradication Programme (GWEP) in the region since being launched in 1989. Only 
10% ascribed natural causes as the cause o f  the malady. Another 10% attributed causation to 
supernatural forces such as witchcraft and Juju. Almost every respondent assigned one 
reason or the other as the cause o f  the disease, w ith only 0.5% claim ing they had no idea 
(Figure 6).
3.2.5 Perceptions of Disease Transmission
A large majority o f the people (66%), felt that Guinea worm was transm itted by “entry into a
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pond by an infected person” . Not withstanding the active nature o f  the GWEP in the area, up 
to 19% o f respondents still claimed they did know the mode o f  transm ission o f  
dracunculiasis. O ther implicated routes o f  transm ission (blood, dirt, sex, and hereditary), 
accounted for 12%. Only 0.5% thought communal eating habits could spread the disease. 
Insect vectors (flies and mosquitoes), were erroneously implicated by 2%  ofthe respondents 
(Figure 7).
3.2.6 Perceptions on Disease Curability
Although there exists no known cure for Guinea Worm (at least for now), respondents 
attributed any remedy that could ameliorate the pain as cure. In this respect, 36% o f  the 
people ranked the use o f  the popular Tamale oil, (an oil formulated by GWEP purposely for 
use in facilitating and hastening worm expulsion) as the most effective cure for Guinea 
worm. As common as it is in most o f  rural Ghana, the use o f  herbs for the “treatment” o f  
Guinea worm was anticipated, with 29% o f the respondents employing herbs for remedies. 
O f those who relied on orthodox drugs, 20%  resorted to the use o f  Paracetamol, with only 
1% actually aware that the drug o f  choice, Ambilhar, could be used to ease the pain and 
hasten worm expulsion. Other methods (worm extraction, injections, going to the hospital, 
use of Maggie cubes, plastering blisters with ant-hill clay), accounted for 10%. Also. 2% 
believed that the disease could be cured/treated by a Juju-man, while 3% had no idea 
concerning cure (Figure 8).
3.2.7 Perceptions on Disease Prevention
Filtration o f pond water before drinking was ranked as the most effective method o f
54
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preventing infection with Guinea worm. Up to 53% o f  the respondents knew this, with 
another 27% aware o f  boiling all drinking water as a preventive measure. The complete 
reliance on Borehole water for drinking to prevent infection was mentioned by 17%. Only 
4% o f the respondents were aware o f  regular use o f  Abate in ponds/dams as a method o f  
breaking transmission (Figure 9).
3.2.8 Source of Knowledge
The respondents were probed to ascertain their sources o f  information/knowledge with 
respect to disease causation, cure, and prevention. A good number o f  them (50%) stated 
Guinea worm workers (coordinators and village volunteers) as their key source o f 
information on preventive remedies. About 20% o f respondents indicated that health workers 
other than the GW personnel were the source o f  their know-how on preventive measures 
(Figure 10).
Assuming the people had a zero knowledge base with respect to prevention before the onset 
o f the GWEP, then it is quite possible the current 15% and 7% levels o f  awareness per 
relations and friends, respectively, could be due to a kind o f  snowball effect where 
information is passed on from person-to-person. This is quite an effective method o f  message 
transmission because o f  its multiplier effect with time. Unfortunately, however, teachers and
religious leaders were the least effective health educators on prevention against the spread o f 
Guinea worm disease, accounting for only 8% and 0.3% respectively.
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56
3.2.9 Respondents’ Principal Sources o f Water
The reliance on stagnant water bodies for water, by most o f  the respondents, was a common 
observation in this study. A vast majority o f  them (73%) depended on ponds/dams for their 
water supplies. Up to 58% o f respondents were observed to depend on pond/dam water all- 
year-round. Also, 36% resorted to the use o f  ponds and dams for water only in the dry season 
(October - May), whilst 5% claimed they made use o f  pond water only during the rains when 
the nearby ponds are filled with water. Only 11% o f  the respondents had access to stand 
pipes as a principal source. Interestingly, this group was observed to be concentrated in 
Kukuo (a suburb o f  Tamale, the regional capital). The 16% o f  respondents who relied 
heavily on borehole water regrettably had to resort to the use o f  pond/dam water in the dry 
season either because the boreholes dried-up, or broke-down due to excessive pressure o f 
usage or both (Figure 11).
3.2.10 Respondents’ Alternative Sources o f Water
In addition to their main sources o f  water supply, inhabitants made use o f  other sources in 
times o f need. These ranged from those who had no alternative source (8%), to a few who 
could access potable water (7%). About 27% turned to boreholes for water as an alternative 
source. The salient observation here was that those who resorted to either standpipe water or
boreholes did so only when the ponds had dried up. This group preferred pond water to 
borehole and/or standpipe water primarily because o f  differences in the taste o f  the water.
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Also, the distance o f  respondents’ home/compound to the source o f  uncontaminated water 
could be a hindrance, and hence, tend to discourage patronage. The second plausible reason 
could be the inconvenience o f  having to form long lines for hours at pipe or borehole sites 
before getting water. Most o f  the people (39%), made use o f  rainwater by trapping and 
storing it for use (Figure 12).
3.2.11 Common Activities At Pond/Dam Sites
The most common activity at the water-contact-site that could aid in the disease transmission 
was wading through the water during the process o f  fetching water. Over 50% o f the people 
drawing water from the ponds/dams did actively wade into the water body before fetching 
water. Understandably, they believed others had contaminated the water at the periphery; 
thus, it is arguably wiser to fetch from the deeper waters.
This practice provides an ideal situation where blisters and/or emerging worms on the skin 
could get into contact with water. This gives the emerging worm a chance to expel larvae 
into the copepod-infested water. The copepod ultimately ingests the larvae and stands the 
chance o f  eventually infecting a person who consumes the water without boiling or filtering, 
unless, o f  course, the pond/dam has Abate added before the water was drawn.
This situation is further aggravated by the fact that majority o f  worms emerge at the lower 
extremities, (the most likely parts o f  the body that comes into contact with water while
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wading). Also, those who engaged in washing clothing and/or cooking utensils at the water 
source (30%), were equally guilty o f  having to wade into the water during their water-related 
activity. Unwittingly, worms at anatomical sites other than the lower extremities also had a 
chance to infect copepods, and therefore sustaining the cycle as demonstrated by the 5% 
swimmers and 14% bathers who had intimate contact with the water source. These are, 
unfortunately activities that are highly implicated in Guinea worm transm ission (Figure 13).
3.2.12 Socio-demographic correlates o f dracunculiasis
To ascertain the dependence o f  Guinea Worm infection on some potential disease-related 
factors, a number o f  hypotheses were tested.
1. Prevalence o f dracunculiasis does not depend on age or level o f education.
Infections with Guinea worm disease were not observed to be age-related (P-value = 0.34). A 
stratified analysis o f  the data, however, implicated some age groups if  the levels o f  education 
were considered as well. Among people with no formal education or only Primary School 
education, infection was age-dependent (P-value = 0.01). Conversely, among people with at 
least, a Senior Secondary School (High School) level o f  education, there was no statistical 
difference in the rate o f  infection within the different age groups. Thus, SSS (P-value =
0.90), and College level education (P-value = 0.63).
Likewise, while within the 10-29 years age group, infection was correlated with level o f  
education (P-value = 0.003), this was not so in the other age groups. Thus, 0-9 years (P-value
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= 0.53), 30-49 (P-value = 0.88), and 50+ years (0.90).
59
2. Prevalence o f dracunculiasis does not depend on occupation.
There is a positive correlation observed between infection with Guinea worm and occupation 
(P-value = 0.04). Hence the disease has a predilection for people with certain occupations. In 
other words, some particular occupations predispose people to infection. Analysis o f  the data 
with respect to the different age groups, however, revealed no relationship between infection 
and occupation within some age groups. Within the 10-29 years age group, infection with 
dracunculiasis did not depend on ones’ occupation (P-value =  0.12). This age group 
constitutes young people who are quite active and mobile. This high rate o f  mobility might 
lessen their level o f  dependency on the contaminated waters.
3. Infection with dracunculiasis does not depend on source o f water.
From these results, it appears that infection with GWD did not only depend solely on the 
individual’s source o f  water, but also, a function o f  both source o f  water and the degree o f  
dependency on this water (Figure 11). Thus, among the investigated sources o f  water supply 
in the communities, only those who collected and depended solely on pond water throughout 
the year contracted infections more often (P-value = 0.002). Those who made use o f  pond 
water but did not rely on this source stood a lesser chance o f  infection (i.e.. pond and stand 
pipe, P-value = 0.03; pond and bore hole, P-value = 0.02; stand pipe only P-value =0.74).
Notably, individuals who made use o f  both ponds and either standpipes or bore holes had a 
high probability o f  contracting infections. This could be due to the fact that, they depend
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mainly on pond water during the dry season when water supply from the utility service is 
often quite erratic and unreliable.
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3.3 DISCUSSION
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The prevalence studies revealed that up to 43% o f the populace still got afflicted with the 
malady during the last transmission season, with 33% suffering it for the second consecutive 
year. This supports the findings o f  Watts (1986), who found that there is no evidence o f 
acquired immunity to infection, and residents in hyper-endemic communities remain 
susceptible to infection all their lives.
The seasonal transmission pattern appears to be an enhancing factor in sustaining the disease 
in the region. Apparently, there is a very long transm ission season, spanning over six months 
with a peak in April to May. This would mean that, any eradication effort should aim at long 
and sustainable methods as against intensive short periods o f  vector control. Any shortfall or 
lapse in control measures would certainly lead to resurgence. This could be one o f the factors 
that militate against the eradication o f  the disease in the region.
It is, however, important to note that the high levels o f  infection among farmers could be 
deceptive. This is more so because that is the main occupation o f  the local people. Likewise, 
the religious predilection or otherwise o f  infected persons could not be ascertained since over 
90% o f the local people in the study area are Muslims.
61
In this study, it was observed that the knowledge base o f  the local people with respect to the 
disease was quite high. Over 50% o f respondents seem to be aware o f  disease causation.
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prevention, and management. This could be due to the efforts o f  the Eradication Program in 
the region. Unfortunately, the status o f  infection is still relatively high (43%). A number o f 
factors could be responsible for this. Obviously, ignorance and poor disease management 
habits on the part o f  some individuals play a very important role in sustaining and 
transmitting the disease. Also, apathy, lack o f  motivation, and worker fatigue could be 
implicated as the major players in promoting the resurgence o f  the disease.
Considering the source o f  knowledge o f  the people with respect to the disease causation, 
prevention and management, one is tempted to implicate the eradication program itself for 
not involving teachers and religious leaders in the eradication effort. These two groups could 
be very effective and reliable means o f  impacting and changing human behavior.
The observation that, infection with Guinea worm is not correlated with age and/or 
educational status is worth noting. This could imply that as one grew older, the level o f  
education no longer plays a role in protection against infection provided the risk factor 
(exposure to infective source o f  water), is still present. This is more so since it is only within 
the 10-29 year age group that there is a negative correlation o f  infection with age. This is 
understandably the youth, who belong to a different era and are more likely to accept and 
adhere to disease prevention measures. Their belief systems are influenced by current events,
and they are more likely to depart from the old-fashioned beliefs o f  their parents which tend 
to maintain the disease transmission.
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63
Not surprising, the children (0-9 years old), indicated no correlation with infection with age. 
This could be attributed to the fact that mothers are most likely not to give their infants pond 
water to drink. They are traditionally given boiled water or depend on breast milk for their 
water requirements.
Chapter 4
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4.0 INVESTIGATION OF WORM MORPHOMETRY
4.1 MATERIALS AND METHODS
A total o f  190 surgically removed gravid female guinea worms collected from various 
districts in the country were used in this study. These are mainly communities that have been 
declared as being endemic for the Guinea Worm Disease (GWD). Specimens were preserved 
in 10% formalin and brought to the laboratory for morphometric measurements. Only whole 
worms (noted to have both anal and buccal regions intact), were used in the studies. No 
fragmented or crushed specimens were used.
4.1.1 Determination of Worm Length
The lengths o f  the worms were measured using a thin inelastic thread. The reference end o f  
the thread was knotted and the appropriate length determined by tracing the thread on the 
entire length o f the specimens. The total length o f  thread so obtained was then stretched out 
on a metre-rule and the approximate length o f  the worm read off. Since the worms are 
purple-white or cream in colour, a black thread was used to produce an appropriate contrast 
for vision (Table 1 and Appendix B).
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4.1.2 Determination of Worm Weight
Having measured the length o f  a worm, its weight was also taken by the use o f  a Mettler 
Teldo (PG 503 ,1996) weighing machine. This is an electronic device that reads up to four (4) 
decimal places. An empty petri dish was placed on the weighing stage and the machine re­
zeroed. The specimens were then placed in the empty petri dish and the weight read (Table 1 
and Appendix B).
4.1.3 Determination of Worm Diameter
Measurements o f  worm diameters were also carried out on a number o f  specimens and 
recorded appropriately. The measurements were done at the mid-sections o f  the worms and 
the average o f  four measurements o f  a given specimen was taken as the approximate 
diameter. These measurements were done, using an Olympus microscope (CH-2; Model- 
CHT, Olympus Optical Co. Ltd, Japan). This was done with the aid o f  a stage micrometer 
and a micrometer eyepiece. The actual measuring was done with the m icrometer eyepiece, 
having calibrated it with the aid o f  the stage micrometer. Readings were taken at a 
magnification o f  40 (Table 1 and Appendix B).
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The mean length o f  the worms was 57.23cm, with the shortest worm measuring 30.5cm and 
the longest specimen measured up to 102.0cm. Considering worm weight, whilst the least 
was 0 .118g, the highest was 2.260g.The mean worm weight was 0.90 lg  (Table 1).
With respect to the morphometric relationships o f  Dracunculus spp., various correlation 
analyses were carried out with the aid o f  computer software - M icrosoft Excel - 1997. Thus, 
the length-age correlation was computed to find out if  there was any correlation between the 
length o f a worm and the age o f  the patient. The computed coefficient o f  correlation was
0.068. Although this is a positive correlation, it shows a very weak association between the 
length o f  a worm and the age o f  the patient. Thus, the length o f  a worm does not in any way 
depend on the age o f  the patient.
Also computed was the weight-age coefficient o f  correlation. This depicted an even weaker 
relationship between the weight o f  a worm and the age o f  its victim. The calculated 
coefficient o f  correlation was 0.03 (about half that o f  the length-age correlation). The overall 
coefficient o f  correlation for weight-length relationship o f  Guinea worm was 0.47, showing a 
weak positive correlation between the weight and length o f  a worm. This revelation could 
mean that the diameter o f  a worm is an important factor w ith respect to its weight. Hence, a 
very long worm could have a small diameter and vice versa. Also, since these worms were
4.2 RESULTS
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surgically removed from the patients, their intrinsic weights could depend on the state/stage 
o f maturity and therefore the numbers as well as the total weight o f  the larvae present in the 
uterus.
Analysis o f  the data based on the sex o f  the patients was carried out. The coefficient o f 
correlation for weight-length relationship o f  worms extracted from males was 0.43 (as 
opposed to 0.47 for both sexes combined). The worm mean length in this case was 57.71cm, 
with a median length o f  57.4cm. Whilst the shortest specimens measured 30.5cm. the longest 
worm was 96.5cm. The mean weight was 0.875g (Table 1).
For specimens extracted from female patients, the weight-length coefficient o f  correlation 
was computed at 0.696 (as compared to 0.43 and 0.47 in the case o f  worms extracted from 
male patients only and for both sexes respectively). The mean, maximum and least worm 
lengths were 59.7cm, 102.0cm and 45.2cm respectively. Likewise, the mean, maximum and 
least worm weights were 0.839g, 1.961 g and 0.602g respectively.
Generally, the mean diameter o f  the worms was computed as 1.32mm. w ith a modal 
thickness o f 1.35mm. The smallest diameter o f  the specimens was 0.75mm. whilst the largest 
worm had a diameter o f 1.96mm.
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From these analyses there appears to exist no relationship between the lengths and weights o f  
Dracunculus spp. on one hand, and the sex o f  the patient. It is however, important to note 
that, there is an apparent correlation, though weak, for weight-length correlation in female 
patients (Correlation Coefficient = 0.696). Generally, however, there was no correlation with 
respect to the sex and age o f  the individual on the one hand, and worm length and/or weight 
on the other hand.
Analyses o f  the relationships between the three morphometric indices o f  the specimens 
(Length, Weight and Diameter), depicts positive correlations for the various paired variables. 
Thus:
1. The weight o f  the worm is associated with the length o f  the worm.
2. The weight o f  the worm is associated with the diameter o f  the worm.
3. The diameter and weight o f  the worm depends on its length.
All the computed coefficients o f  correlation were positive. The Length-Diameter Coefficient 
o f Correlation was observed to be the least (0.653). On the other hand, the Coefficient o f  
Correlation for Weight-Length and Weight- D iameter were 0.779 and 0.812 respectively.
4.3 DISCUSSIONS AND CONCLUSIONS
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From these analyses, it can be inferred that these three morphometric parameters are 
positively correlated. It is also important to note the positively strong correlation shown by 
the length-weight (0.779) and diameter-weight (0.812) variables. These relationships do not 
necessarily imply causation. Hence, it does not necessarily mean that the weight o f  a worm 
depends solely on its diameter and vice versa. Likewise, the weight o f  a worm cannot be said 
to depend on its length only.
Also, it is important to note that, the exact ages o f  these worms were unknown. Hence, it 
cannot be known whether worms recovered from women were more likely to be older and 
therefore more mature and/or larger/heavier.
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5.0 HISTOMORPHOLOGY OF DRACUNCULUS MEDINENSIS
5.1 MATERIALS AND METHODS
5.1.1 Tissue Preparation
Tissue preparations were carried out on alcohol-preserved specimens for histologic details. 
Microscopic examinations were carried out on sections (T/S) o f  the mature female worms 
(Figures 14 -  21). The specimens were first thoroughly rinsed with tap water. Pieces 
measuring 1.0 - 1.5cm long were then cut from the anterior, pharyngeal, mid and posterior 
regions o f the specimens for serial sectioning. The cut pieces were washed in 10% Potassium 
Hydroxide (KOH) for 4-5 minutes. These were then rinsed with tap water and dehydrated at 
room temperature (29 °C) by passing it through a series o f  progressively more concentrated 
alcohol baths (i.e. H20  —>30 —> 50 —> 70 —* 95% —* absolute alcohol) for 45 m inu ts each. 
Placing in xylene briefly then cleared the dehydrated specimens.
The cleared pieces o f  worm were then impregnated with paraffin/candle wax for 24 hours at 
a temperature o f  56°C. These wax-impregnated specimens were then imbedded in blocks o f  
candle wax measuring 1cm by 1cm by 2cm each. Sections were then cut out using a large 
rotatory microtome (LR - 85. OSK. - 9782; Yamato Kohki Industrial Co. Ltd, Japan). The 8-
Chapter 5
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71
micron (8|J.) sections obtained were dewaxed/cleared in xylene and again passed through an 
alcohol series (i.e. absolute —> 96 —> 70 —> 50 —■> 30% alcohol) to re-hydrate. The sections 
were over-stained in Borax Carmine and differentiated briefly in acid-alcohol before counter- 
staining in eosin. The sections were washed in 70% alcohol after each staining. After counter 
staining in Eosin, the preparations were again dehydrated briefly in 96% and absolute alcohol 
respectively and cleared in xylene. The preparations were mounted in dpx and examined 
under a microscope for morphologic details.
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The histomorphology o f  Dracunculus medinensis (the anatomical composition o f  the 
worm), was observed to vary along the anterio-posterior axis. There is a progressive change 
in anatomy anterio-posterialy. As typical o f  all nematodes, the outer covering o f  the worm is 
a tough cuticle. The cuticle was found to be multi-layered, consisting o f  a cortical, a  medial 
and a basal layer. A well-developed hypodermis, lying between the cuticle and the muscle 
layer was observed. The hypodermis is thickened in the dorsal and ventral positions to form 
the hypodermal cords. The muscle cells are the same from the anterior to the posterior 
regions, although the number and shape vary. They overlap, and depending on the region, 
they may look large or small (Figures 1 4 -2 1 ) .
5.2.1 The Cephalic Region
The anterior region was observed to be quite unique. The mouth is triangular in shape and 
leads into the esophagus, which has a trivoliate, cuticle-lined lumen. The contractile portions 
of the buccal musculature are well developed and distinct. The non-contractile segments o f 
the longitudinal muscles are however not well differentiated in this region. There are no 
larvae, implying that the uterus does not extend to this segment o f  the worm (Figure 14).
5.2 RESULTS
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5.2.2 The Pharyngeal Region
The esophagus is round, but assumes an oval shape towards the upper mid-section o f  the 
worm. The muscle cells o f  the pharyngeal region are well differentiated. Some cells o f  the 
non-contractile muscle cells are observed to be nucleated. The esophagus is perpendicularly 
oriented to the pseudocoel and lies along the axis o f  the lateral nerve cords. The syncytial 
hypodermis is also well differentiated here (Figure 15 and 16).
5.2.3 The Mid-Section
The shape at the pharyngeal region is completely modified or differentiated into a circular 
shape at the mid-section. The pseudocoel here is almost circular and its diameter is 
considerably increased. There are two bean-shaped identical layers o f  muscles cells that lie 
opposite each other. These are separated at either side by the lateral cords where the 
hypodermis projects into the body cavity. The thickness o f  the bean-shaped layer o f  muscle 
cells progressively decreases from the mid section to the posterior region. The gut is often 
difficult to see, and almost always displaced by the larvae-filled uterus, and pushed to one 
side o f the body cavity. It is apparently free o f  any contents. The longitudinal muscle cells 
are well developed in this region. However, the non-contractile parts o f  the muscle cells are 
flattened against the contractile portions and not easily seen (Figure 17 - 20).
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5.2.4 The Posterior Section
The characteristic circular shape at the mid-section is maintained at the posterior end o f  the 
worm. The thickness o f  the two bean-shaped muscles is, however, considerably reduced. The 
larvae filled uterus is still the prominent organelle in this region. The gut is however, 
markedly reduced to an orifice (Figure 21).
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5.3 DISCUSSIONS AND CONCLUSIONS
At maturity, the gut o f  the female guinea worm is completely atrophied and the entire worm 
is completely made-up o f  the larvae-filled uterus. From this study, differences in the 
musculature from the anterior region, with much thicker muscles to the mid region was 
observed. The thicker muscles could be used for squeezing the larvae out into water by 
contractions. Thus, by intermittent active expulsions o f  larvae when exposed segment o f  
worm or blister comes into contact with water. It appears the stimulus for contraction is 
provided by cold water. It has also been observed that the number o f  larvae expelled 
progressively decreases, with the highest coming from the first immersion/contact (Chandler, 
et al., 1961). This contention is further buttressed by the fact that protruding/exposed 
segment o f the worm dries up, and therefore is non-functional. This could account for the 
reducing numbers in the larvae expelled per immersion in water.
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6.0 VECTOR SPECIES OF DRA CUNCUL US MEDINENSIS
6.1 MATERIALS AND METHODS
6.1.1 Sampling
Copepods (Figure 2), were collected form ponds/dams often used as sources o f  water. The 
collected water from these water bodies was observed to be used mostly for drinking (at 
home and on the farms), as well as for other domestic purposes. Samples were collected 
during peak seasons o f  the disease for two seasons. November 1998 to May 1999, and again 
from November 1999 to May 2000.
In sampling, a wide mouthed 4-liter plastic container was lowered into the water at the 
contact site and scooped. The water was filtered through a monofilament filter o f  70|o.m 
mesh. The copepods so trapped were washed into a beaker. This was repeated four times and 
the collected samples fixed immediately in 10% formalin and brought to the laboratory for 
identification.
6.1.2 Identification
Copepods were identified using the key prepared by Olsen (1993). A phase-contrast 
microscope (Model: Optiphot-2; Nikon, Japan) was used. Only mature females (noted to be
Chapter 6
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77
carrying egg sacs), were examined and identified accordingly. The use o f  this key was 
supplemented by another key prepared by Boxshall and Braide (1991). The two keys use 
distinct morphological features on such parts o f  the body as the fifth pair o f  legs, the caudal 
rami, the antennae, the seminal receptacle, the maxillary palpi, the total body length and the 
relative lengths o f  the body segments.
The identification process involved handling the specimen in water-free glycerine on a clean 
slide under a microscope. The copepod was then teased with a dissecting pin until the 
structure under investigation was appropriately oriented for observation. The laboratory-bred 
copepods were identified before being evaluated for their infection potential.
6.1.3 Colony Maintenance
Specific cyclops species were reared in 500ml beakers in the laboratory at room temperature 
(26 - 29 °C). First, a single gravid female copepod was selected using a Pasteur pipette and 
washed several times with tap water in a  petri-dish. The petri-dishes were placed over a dark 
background to facilitate easy access, handling and observation o f  the copepods. This gravid 
female was then transferred into a beaker containing 500ml tap water. The nauplii hatching 
from the eggs were fed on cow dung infusion and the green algae Cladophora spp. The water 
in the beakers was aerated for 5 minutes daily and changed every fortnight until the colony 
could provide enough adult copepods for infection with Guinea worm larvae.
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6.1.4 Evaluation of Infection Potentials
Vectors o f  D. medinensis have hitherto been identified by elucidating the species o f  
copepods (sampled from domestic water sources), which are noted to be naturally infected 
with Guinea worm larvae. As Yelifari (1997), postulates, the high mobility o f  the local 
people during the dracunculiasis transm ission season presupposes that some o f  them could 
have been infected by water sources other than the ones investigated. It is therefore difficult 
to correlate occurrence o f  cyclopoid copepods in the local water sources with the prevalence 
o f human infection in the catchment area. Hence, no valid conclusions as to the most 
important intermediate host(s) in these areas can often be drawn.
In this study, laboratory experiments were carried out to infect various species o f  copepods 
with first stage (L,) guinea worm larvae (Figure 22). The larvae were obtained from matured 
female worms extracted from patients. Only pre-emergent whole worms were used in this 
study. The worms were extracted from patients by trained worm extractors after 
administering local anesthesia (Figure 24 - 28). To ensure viability o f  the larvae, the worms 
were immediately milked after extraction into filtered pond water. In addition to the tests on 
the laboratory-reared copepods, specimens obtained from the field were also tested.
Using a Pasteur pipette, 1 -2 drops o f  the larvae-filled milky fluid were introduced into 500ml 
beakers containing 50 copepods o f  each species. Preliminary' infectivity tests with composite 
samples showed that varying numbers o f  larvae could be ingested by different species and
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different developmental stages o f  the vectors. These composite samples contained a mixture
o f copepods species sampled from the field, with various developmental stages o f  the 
vectors. Hence, the experimental set up was categorized into adults and copepodids. Each 
species had three replicates (adults only, copepodids only, and a m ixture o f  adults and 
copepodids, each with a control. In this experiments, the following attributes were evaluated.
1. The ability o f  the various species o f  copepods to ingest the first stage guinea worm 
larvae (Figure 22).
2. The maximum number o f  larvae that adults and copepodids could ingest.
3. How long each developmental stage o f  copepods used in the experiment could stay 
alive with respect to the number o f  larvae ingested.
4. The ability o f  ingested larvae to develop from L, to the infective L3 (Figure 23), in 
each species.
Samples o f  the “infected” copepods were observed under a m icroscope progressively (after 
12, 24, 48, 72, hours; and 7, 10, 14, 21, 28 days etc), for the presence and viability o f  
ingested larvae. A drop o f  ice water was put on the Cyclops on a m icroscope slide to 
immobilize it, leaving the active larvae that could then be seen in the haemocoel o f  the 
copepod. Observations for copepod mortalities were recorded accordingly.
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6.2.1 Copepod Species
A total o f  7 copepod species (4 Thermocyclops and 3 Mesocyclops spp.) were found in the 
study. The most common species in the study communities was M. keiferi. Whilst M  keiferi 
was present in all 5 communities, M. aspericornis, T. incisus, and T. inopinus were present in 
3 communities each. Also, T. oblongatus was present in 2 communities, with T. neglectus 
andM  major occurring in only one community each. Table 2 shows a summary o f  copepod 
species found in the study area.
6.2.2 Infection Potentials
Four o f the copepod species were observed to be capable o f  supporting development o f 
guinea worm larvae (Table 3). All 4 species o f  copepods were exposed to first stage-larvae 
(L|) o f guinea worm. The number o f  larvae ingested as well as the ability o f  the copepod to 
stay alive and facilitate the development o f  the first-stage larvae into the infective third-stage 
larvae was evaluated. Among the adult copepods the larger Mesocyclops spp. were observed 
to be the most voracious. Up to 4 larvae could be ingested by a single copepod. Conversely, 
the smaller Thermocyclops spp. could ingest a maximum o f only 2 - 3  larvae (Table 3).
Like the adults, the copepodid stages o f  the copepods did ingest the larvae. However, these 
could not ingest more than 2 larvae per copepodid (except the juveniles o f  M. keiferi).
6.2 RESULTS
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Among both the adults and juveniles, infected copepods that were able to withstand the 
infestation for 72 hours (3 days), were observed to survive the infection. Also, in all 4 
species o f  copepods, the larvae were able to moult and therefore to develop to the infective 
L3 stage within 14-20 days (Table 3).
Although all copepod species studied did ingest Guinea worm larvae, very high mortalities 
were observed among the adults. M. aspericornis and M. keiferi ingested up to 5 larvae (81 
and 75% respectively). However, by the third day (72 hours), only 12 and 6% o f the infected 
copepods were still alive (i.e., 88 and 94% mortalities occurring). The smaller 
Thermocyclops spp. could ingest only between 2 and 3 larvae, with a corresponding 
percentage mortalities o f  4 and 0% for T. incisus and T. oblongatus respectively (Table 3).
With respect to the copepodids, the maximum number o f  larvae ingested was 2. No 
remarkable mortalities were observed within the 72-hour critical period. The 2% mortality 
recorded was attributed to incidental death (Table 3).
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O f all the 7 cyclops species found in the study area, the most common species was M. keiferi. 
In order o f  relative importance, therefore, M. keiferi —> M. aspericornis —> T. incisus —>
T. inopinus —> T. oblongatus —> T. neglectus —>M  major. All but M. aspericornis have
been found by a number o f  researchers to be naturally infected with Guinea worm larvae. 
Yelifari et a l, (1997), found T. incisus, T. inopinus, and M. keiferi sampled from water 
bodies in the Tamale municipality to be infected with Guinea worm larvae.
The adults o f  the relatively larger species (mainly Mesocyclops spp.), recorded very high 
mortality rates upon infection with the first stage larvae (L.) o f  the parasite. The highest 
copepod mortality rate was recorded by M. keiferi (94%). However, the copepodid stages o f  
these species were able to withstand infection for extremely longer periods. The smaller 
genera (mainly Thermocyclops spp.), did not record any remarkable mortalities on ingesting 
parasite larvae (L,). The copepodid stages ingested mostly one larva each. This was 
comparable to the scenario in the Mesocyclops spp (Table 3).
The very high mortalities encountered in adults o f  the larger Mesocyclops spp. could be 
attributed to the fact that the ingested larvae draw nutrients from the copepods. Thus, hyper­
infestation could lead to physiological upsets in the host as a result o f  the feeding regime o f
6.3 DISCUSSION
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the ingested larvae. This could further explain the observation by some researchers that, the 
ingested larvae feed on the ovaries o f  the copepod. What might actually be happening is that, 
by drawing nutrients form the copepod, its ovaries become atrophied and non-functional as a 
result o f  the physiological upset. Also, the development o f the ingested first- stage larvae is 
observed to be faster in the adult copepods than in the copepodids. This could be due to the 
availability o f  nutrients, and the adult copepods have a greater reserve o f  nutrients upon 
which to draw. Also, the copepodid stages are themselves growing and thus require high 
nutrient amounts. In this fashion, development from the first stage larvae to the infective 
third stage larvae in adult and juvenile copepods infected at the same point in time definitely 
go out o f  phase, with larvae in adult vectors reaching the infective stage earlier than in the 
copepodids.
This apparent lag in development could result in differential manifestations o f  the disease 
(especially, the peak season), from year to year in the same locality. This becomes quite 
explicit, depending on the vector species that dominates in the community. This assertion is 
further buttressed by the fact that, usually in each endemic zone only one o f  the local 
predatory species o f  copepods is often found to be the dominant intermediate host by virtue 
o f its preferred habitat, its seasonal population dynamics, or both (Onabamiro, 1954; 
McCullough, 1982).
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The most important vectors o f  dracunculiasis in the study areas are: M. keiferi —> M. 
aspericornis —> T. incisus —> T. inopinus —> T. oblongatus . However, it is important to
note that, the copepodids could be the main players in the transm ission cycle since adults die 
massively (70-90%) upon ingesting the first stage larvae o f  the parasite. In the 
Thermocyclops spp, both adult and juvenile stages were observed to be equally efficient in 
habouring the larvae for the required periods needed for it to develop to the infective L3 
stage.
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Currently, the taxonomy and species classification o f  Dracunculid parasites known to infect 
both humans and other animals is based on type o f  host and morphological features o f  the 
parasite. The morphological markers are sufficient for systematic analysis o f  these parasites, 
however, most o f  the key features are present on the male worms, which are rarely, i f  ever, 
available for study. Therefore, these probably need to be supported by molecular 
characterization. Employing molecular probes, polymerase chain reaction amplification, and 
sequence-based identification is therefore, advantageous for substantiating epidemiologic 
relationships and diversity. Since this DNA speciation can be accomplished even on small 
portions o f  extracted female worms, it lends itself to taxonomic characterization.
As the number o f  genotypes o f  Dracunculus spp. responsible for human infections is still 
unknown, determining whether the epidemic under investigation is caused by multiple 
strains or a single species is often a dilemma. This makes it difficult to ascertain the 
source(s) o f infections, and therefore does not allow for the formulation o f needed prevention 
and control guidelines. Hence, there is the need for strain characterization o f  Dracunculus 
spp. for epidemiologic tracking (Lai and Tibayrenc, 1997).
Chapter 7
7.0 MOLECULAR EPIDEMIOLOGY OF GUINEA WORM
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The role o f  zoonotic infections o f  Dracunculid parasites in human infections is also yet to be 
ascertained. Thus, the species types and genotyping tools are needed for studies o f the 
molecular epidemiology o f  dracunculiasis. Such tools would facilitate laboratory 
characterization o f  dracunculiasis cases and identification o f  infection sources. The 
variations or otherwise in genotypes may also shed light on the transmission dynamics o f  the 
Dracunculid parasites in different geographic areas and epidemiologic settings. An 
intragenotypic heterogeneity in the SSU-rRNA sequence by isolates could be a very useful 
tool for epidemiologic investigations.
The occurrence o f  multiple genotypes at the same geographic location or the distribution 
patterns o f these genotypes would define the complexity o f  Guinea worm epidemiology. 
Divergence or aggregations o f  isolates from various geographic locations (countries) might 
confirm or dispel the idea o f localized transmission cycles. Claims and allegations o f  
imported cases could therefore be investigated and the true origins ascertained.
In this study, we report on the use o f  sequence data from the SSU-rRNA and the Internal 
Transcribed Spacers land 2 (ITS1 and ITS2) to distinguish among the species o f Guinea 
worm. This represents a first step to determine genetic variation within isolates from 
specimens collected from humans and from some identified animals o f  various geographical 
locations. Relationship among species was inferred from DNA sequence data. Phylogenetic 
inference among Dracunculus species was done by examining a conserved region o f  the
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complete gene sequence o f  the genomic DNA (the gene sequence o f  18S-rRNA), and 
subsequently, the internal transcribed spacer 1 -1TS1 and ITS2 regions for sub-species 
(Figures 29 and 30).
DNA sequences o f  isolates collected from various geographical locations (mostly in Africa) 
were analyzed for sequence variability. In order to investigate the possibility o f  more than 
one species o f  Dracunculus in humans, Guinea worms originating from different African 
countries, Pakistan and Yemen were compared by sequence analysis o f  the nuclear small 
subunit 18S rRNA gene as well as the more variable ITS 1 and ITS2 regions.
Since the highly conserved regions in the ribosomal repeat array can be used for study o f 
relationships across phyla (Gerbi, 1985: as cited in Lai and Tibayrenc. 1997), the more 
variable ITS-1 and ITS-2 regions was targeted to investigate for sub-species (a lower 
taxonomic resolution/level). This is because the ITS regions does not encode for any product, 
permitting it to evolve at a faster rate than the ribosomal coding regions. The level o f 
variation in this region therefore makes it suitable for detecting genetic diversity.
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7.1 MATERIALS AND METHODS
7.1.1 Worm Collection
The specimens (Guinea worms), were collected mainly from villages in Northern Ghana, 
declared as endemic for dracunculiasis. Specimens from other African countries (Cameron, 
Niger. Nigeria, Togo, Sudan, Cote d ’Ivoire, Ethiopia, and Burkina Faso) were also used in 
the investigation. Outside o f the African continent, specimens from Pakistan and Yemen 
were used (Figure 31). These specimens were preserved in 10% alcohol (a few from Ghana 
were kept frozen). All specimens were pre-emergent worms extracted from patients (Figure 
25). Two interesting cases o f  a “Red” worm (from Togo), and a worm o f animal origin (from 
a dog in Ghana) were also used in the study.
7.1.2 DNA Extraction and Preparation
DNA was extracted from specimens following the procedure outlined in - “ DNA Extraction 
from Animal Tissue” as per “Bio 101 FastDNA Kit” (Applied Biosystems: Appendix C). 
The extracted DNA was then purified using QIAquick kit (Appendix D). to remove PCR 
inhibitors.
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7.1.3 Polymerase Chain Reaction (PCR) Amplification
To analyze the base sequence for speciation, the 18S rRNA region o f  the rRNA cluster was 
targeted (Figure 31). Extracted DNA was amplified using the forward primerNEMFG1 (5’ 
TCT CCG ATT GAT TCT GTC GGC GAT TAT ATG), and the reverse primer CRYPTOR 
(5’ GCT TGA TCC TTC TGC AGG TTC ACC TAC).
To type the isolates, however, the Internal Transcribed Spacer Region 1 (ITS 1) was targeted. 
Here, DNA was amplified using the forward primer NAP9 (AAC AGG TCT GTG ATG 
CCC T), and the reverse primers 58S-R1 (TAG CTG CGT TCT TCA TCG ATC), 58S-R2 
(TTG CTG CGT TCT TCA TCG ATC), and 58S-R3 (TAG CTG CGT TCT TCA TCG 
ACC).
PCR reaction mixtures consisted o f  IX  buffer, (10 mM Tris-HCl, 50mM KC1, 1.5mM 
MgCl2, pH 9.0), 200p.M o f dNTP, 25pmol o f  each primer. 2.5 U o f  AmpliTaq Gold (Perkin 
Elmer), and 0.1-0.5|il o f  DNA, in each final volume o f  50pl. Forty-five PCR cycles (94 °C 
for 30 seconds, 60 °C for 30 seconds, 72 °C for 90seconds) were carried out in an automated 
Thermal Cycler GeneAmp Perkin-Elmer 9700 with an initial hot start o f  95 °C for 15 
minutes, and a final extension at 72 °C for 10 minutes.
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7.1.4 Verification o f PCR Product
To visualize the products, the PCR products were run through 1% Agarose gel in TBE (gel 
electrophoresis), which was subsequently stained with Ethidium Bromide to ascertain DNA 
amplification or otherwise. A lOObp ladder was used as the standard (Figures 32 - 34).
7.1.5 DNA Sequencing
Amplification products were purified using spin columns (Stratagene kit), according to the 
instructions from the manufacturer (Appendix E), and eluted in 50p.l o f UV-H-,0. Sequencing 
reactions for speciation were performed with the purified products on both strands using the 
primers NEMFG1 and CRYPTOR, as well as the internal primers 5 ’ CTG CCT TAT CAA 
CTT TCG ATG (NEM1F), 5 ’ CAT CGA AAG TTG ATA AGG CAG (NEM 1R), 5 ' GCG 
GTT AAA AAG CTC GTA GTT GG (NEM2F), 5 ’ CCA ACT ACG AGC TTT TTA ACC 
GC (NEM2R), 5 ’ GCG GCT TAA TTT GAC TCA ACA C (NEM3F), 5 ' GTG TTG AGT 
CAA ATT AAG CCG C (NEM3R), 5 ’ CCG GGA CTG AGC CGT TTC GAG (NEM4F), 
and 5’ CTC GAA ACG GCT CAG TCC CGG (NEM4R). However, sequencing reactions for 
typing isolates were performed with the purified products on both strands using the same 
primers employed in DNA amplification.
The products were purified with CENTRl-SEP Protocol (Applied Biosystems), to remove 
Dye Terminators prior to sequencing (Appendix F). The products were then dried w ith a 
DNA Speed Vac (Model 120, ThermoSavant), and re-suspended in acrylamide. Sequencing
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was carried out with a 3100 Genetic Analyzer (Applied Biosystems). The base sequences 
were assembled and analyzed using Seqman.
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The gene coding o f  isolates o f  human origin (D. medinensis) from the various locations were 
successfully extracted, amplified and sequenced. The Small Subunit Ribosomal RNA (SSU- 
rRNA) o f  these isolates was constructed/assembled (Appendix G).
All the isolates were observed to have identical sequences. PCR amplification o f  small 
subunit ribosomal (SSU-rRNA) gave a single product from each species about 1819 base- 
pairs long. Figure 32 -  34) illustrates an electrophoretic analysis (in 1% agarose gel) o f  PCR- 
amplified products o f  guinea worm SSU-rRNA.
Interestingly, the gene coding o f  an isolate o f  a worm o f  animal origin (a dog from Ghana), 
revealed an identical base sequence to sequences o f  isolates from worms extracted from 
humans. This implies that D. medinensis has low host specificity and can infect animals 
other than man. The “Red” worm o f human origin (Togo) produced a sim ilar base sequence 
as those o f normal coloration (pale-white).
These results are quite interesting in relation to potential incidentalanimal infections with 
Guinea worm in endemic communities. The identical base sequences o f  the “Dog Guinea 
worm” to those extracted from humans implies that, the disease can be transmitted from man 
to dogs and vice versa. This is not a welcome revelation, especially at this point in time when 
global eradication efforts are intensified. This is more so because it is possible to distinguish 
D. insignis from D. medinensis at the SSU-rRNA locus.
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7.2 RESULTS
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Fortunately, a more suitable reverse primer from the base sequences obtained from the ITS 
studies was designed. This primer does not produce the undesirable problems such as the 
formation o f  dimmers that we had to contend with when we had to use CRYPTOR as the 
reverse primer. This primer is 18SR(GTT AAT GAT CCTTCC GCA GGTTCA CCT AC).
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1. Dracunculus medinensis and D. insignis can be differentiated by the SSU-rRNA 
sequence. This may apply as well to other Dracunculus isolates from animals.
2. All Dracunculus medinensis isolates have the same sequence o f  the SSU-rRNA.
3. All the ITS 1 sequences look identical, including the Dog isolate from Ghana (o f course, 
it would have been more interesting if we found differences).
4. Thus, strain typing has to be done using other regions, if  possible, to answer the question 
as to whether or not some animal isolates are really different from the human isolates.
5. Definitely, species that do not infect humans like Dracunculus insignis may be 
differentiated on the basis o f  the SSU rRNA.
6. It is possible to verify whether or not worms o f  animal origin are D. medinensis or 
otherwise based on the 18S (SSU rRNA) and the ITS1 regions.
7. IMPLICATIONS: Guinea worm can possibly be transmitted from man to animals 
(dogs), and vice versa.
7.3 CONCLUSIONS
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Chapter 8
8.0 GENERAL DISCUSSION
In this study, it was found out that a considerably high percentage o f  the local people in the 
study communities could still fall prey to the guinea worm disease, even though there has 
been an active eradication programme in the area for over a decade. The evaluated incidence 
for the last transmission season was 43%, with 33% suffering from the disease for the second 
consecutive year. This collaborates the findings o f  Watts (1986) that there is no evidence o f  
immunity to infection, and residents in hyper-endemic communities could remain susceptible 
to infection all their lives.
The seasonal transmission pattern appears to be a major factor in sustaining the disease in 
endemic communities. This could be due to the very long transm ission seasons in some 
endemic communities. This would imply that, any eradication effort should aim at long and 
sustainable methods as against intensive short periods o f  vector control. Any shortfall or 
lapse in control measures would certainly lead to resurgence. This could be one o f  the factors 
that militate against the eradication o f  the disease in the region.
The observed high infection rate among farmers could be deceptive. This is because farming 
is the main occupation o f  the local people. Likewise, the religious predilection or otherwise 
of infected persons could not be evaluated because over 90% o f  the people in the study area 
are Muslims.
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In this study, it was observed that the knowledge base o f  the local people with respect to the 
disease is quite high. Over 50% o f respondents seem to be aware o f  disease causation, 
prevention, and management. This could be due to the efforts o f  the Eradication Program in 
the region. Unfortunately, the status o f  infection is still relatively high (43%). A number o f  
factors could be responsible for this. Obviously, reluctance o f  the local people to comply 
with disease management and control practices could play a very important role in sustaining 
and transmitting the disease.
Considering the source o f  knowledge o f  the people with respect to the disease causation, 
prevention and management, one is tempted to implicate the eradication program itself for 
not involving teachers and religious leaders in the eradication effort. These two groups could 
act as very effective and reliable means o f  impacting and changing human behavior. It is 
therefore suggested that the GWEP in the country should co-opt these opinion leaders to 
intensity the eradication effort.
The observation that, infection with Guinea worm is not correlated with age and/or 
educational status is worth noting. This could imply that as one grew older, the level o f  
education no longer plays a role in protection against infection provided the risk factor 
(exposure to infective source o f  water), is still present. This is more so since it is only within 
the 10-29 year age group that there is a negative correlation o f  infection with age. This is 
understandably the youth, who belong to a different era and are more likely to accept and
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adhere to disease prevention measures. Their belief systems are influenced by current events, 
and they are more likely to depart from the old-fashioned beliefs o f  their parents, which tend 
to maintain the disease transmission.
Not surprisingly, the children (5-9 years old), indicated no correlation with infection with 
age. This could be attributed to the fact that this age group is likely to heed to instructions 
and advice from their parents, and therefore not drink from ponds.
The morphometric analysis o f  specimens for possible correlations revealed that there appears 
to exist no relationship between the lengths and weights o f  Dracunculus spp. on one hand, 
and the sex o f  the patient. The apparent correlation, though weak, for weight-length 
correlation in female patients (Correlation Coefficient =  0.696) could be due to hormonal 
factors on the part o f  the human host. Generally, however, there was no correlation with 
respect to the sex and age o f the individual on the one hand, and worm length and/or weight 
on the other hand.
All the computed coefficients o f  correlation were positive. The Length-Diameter Coefficient 
of Correlation was observed to be the least (0.653). On the other hand, the Coefficient o f  
Correlation for Weight-Length and Weight- Diameter were 0.779 and 0.812 respectively. It 
is suggested that an animal model be employed to investigate this further, since the parasite 
does not avail itself for scientific investigation in the human host due to the very long pre­
patent period.
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It can be inferred that these morphometric parameters are positively correlated. It is also 
important to note that the strong positive correlation shown by the length-weight (0.779) and 
diameter-weight (0.812) variables may not necessarily imply causation. Also, it is important 
to note that, the exact ages o f  these worms were unknown. Hence, it cannot be known 
whether worms recovered from women were more likely to be older and therefore more 
mature and/or larger/heavier.
This observation is further buttressed by the observation that, at maturity, the gut o f  the 
female guinea worm is completely atrophied and the entire worm is completely made-up o f 
the larvae-filled uterus. The observed differences in the musculature from the anterior region, 
with much thicker muscles to the mid region could be an adaptation to enable the mature 
worm to expel, its larvae into water by muscular contractions. Thus, by intermittent active 
expulsions o f  larvae when exposed segment o f  worm or blister comes into contact with 
water. It appears the stimulus for contraction is provided by water. It has also been observed 
that the number o f  larvae expelled progressively decreases, with the highest coming from the 
first immersion/contact (Chandler, etal., 1961). This contention is further buttressed by the 
fact that protruding/exposed segment o f  the worm dries up, and therefore is non-functional. 
This could account for the reducing numbers in the larvae expelled per immersion in water. It 
is the deposition o f  larvae into domestic water sources containing the appropriate vector 
species o f the parasite, which ensures the transm ission o f  the disease.
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In this study, seven Cyclops species were found in the study communities. The most 
common species was M. keiferi. Ranking these gives the following in order o f  relative 
importance: M  keiferi —> M. aspericornis —> T. incisus —> T. inopinus —> T. oblongatus
—> T. neglectus —>M. major. Among these, all but M. aspericornis have been found by a
number o f  researchers to be naturally infected with Guinea worm larvae. Yelifari et a l,
(1997), found T. incisus, T. inopinus, and M. keiferi sampled from water bodies in one o f  the 
study localities (Tamale) to be naturally infected with Guinea worm larvae.
It was observed that, the adults o f  the relatively larger species (mainly Mesocyclops spp.), 
recorded very high mortality rates upon infection with the first stage larvae (L,) o f  the 
parasite. The highest copepod mortality rate was recorded by M. keiferi (94%). However, the 
copepodid stages o f  these species were able to withstand infection for extremely longer 
periods. The smaller genera (mainly Thermocyclops spp.). did not record any remarkable 
mortalities on ingesting parasite larvae (L,). The copepodid stages ingested mostly one larva 
each. The very high mortalities encountered in adults o f  the larger Mesocyclops spp. could be 
attributed to the fact that the ingested larvae draw nutrients from the copepods. Thus, hyper­
infestation could lead to physiological upsets in the host as a result o f  the feeding regime o f 
the ingested larvae. What might actually be happening is that, by drawing nutrients form the 
copepod, its ovaries become atrophied and non-functional as a result o f  the physiological 
upset. Also, the development o f  the ingested first- stage larvae is observed to be faster in the
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adult copepods than in the copepodids. This could be due to the availability o f  nutrients, 
since the adult copepods have a greater reserve o f  nutrients upon which to draw. Also, the 
copepodid stages are themselves growing and thus require high nutrient amounts. In this 
fashion, development from the first stage larvae to the infective third stage larvae in adult 
and juvenile copepods infected at the same point in time definitely go out o f  phase, with 
larvae in adult vectors reaching the infective stage earlier than in the copepodids.
This apparent lag in development could result in differential manifestations o f  the disease 
(especially, the peak season), from year to year in the same locality. This becomes quite 
explicit, depending on the vector species that dominates in the community. This assertion is 
further buttressed by the fact that, usually in each endemic zone only one o f  the local 
predatory species o f  copepods is often found to be the dominant intermediate host by virtue 
o f its preferred habitat, its seasonal population dynamics, or both (Onabamiro, 1954; 
McCullough, 1982).
It is, however, important to note that, the copepodids could be the main players in the 
transmission cycle since adults die massively (70-90%) upon ingesting the first stage larvae 
of the parasite as a result o f hyper-infestations. In the Thermocyclops spp, both adult and 
juvenile stages were observed to be equally efficient in habouring the larvae for complete 
development to the infective stage.
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Studies to ascertain the molecular epidemiology o f  Dracunculus species revealed that:
1. Dracunculus medinensis and D. insignis can be differentiated by the SSU-rRNA sequence. 
This may apply as well to other Dracunculus isolates from animals.
2. All Dracunculus medinensis isolates have the same sequence o f  the SSU-rRNA.
3. ITS 1 sequences o f  isolates from different geographical areas look identical, including the 
Dog isolate from Ghana. O f course, it would have been more interesting if we found 
differences.
4. Strain typing has to be done using other regions, if  possible, to answer the question as to 
whether or not some animal isolates are really different from the human isolates.
5. Definitely, species that do not infect humans like Dracunculus insignis may be differentiated 
on the basis o f the SSU rRNA.
6. It is possible to verify whether or not worms o f  animal origin are D. medinensis or otherwise 
based on the I8S (SSU rRNA) and the ITS1 regions.
7. This may imply that Guinea worm can possibly be transmitted from man to animals (dogs), 
and vice versa.
It is therefore recommended that further studies be carried out using suitable animal models to 
find out whether or not D. medinensis is capable o f  successful development in some animals.
101
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102
TABLES
TABLE 1: WORM MORPHOMETRY
PARAMETER
MEAN SMALLEST LARGEST
ALL WORMS
Length (cm) 57.2 30.5 102.0
Weight (g) 0.901 0.118 2.260
WORMS FROM MALES
Length (cm) 57.71 30.5 96.5
Weight (g) 0.875 0.118 2.260
WORMS FROM FEMALES
Length (cm) 59.7 45.2 102.0
Weight (g) 0.839 0.602 1.961
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103
TABLE 2: CYCLOPOID COPEPODS FOUND IN THE STUDY AREA
COMMUNITY COPEPOD SPECIES FOUND
1998/99 SEASON 1999/2000 SEASON
T. inopinus T. inopinus
NYANKPALA M. kieferi T. neglectus
M. aspericornis M. kieferi 
M. aspericornis
T. incisus T. incisus
DIARE T. oblongatus T. oblongatus
M. kieferi M. kieferi
SAVELUGU T. incisus T. incisus
M. kieferi M. kieferi 
T. oblongatus
T. inopinus, T. inopinus,
KUKUO T. incisus, T. incisus,
M. kieferi 
M. aspericornis
M. kieferi
T. inopinus. T. inopinus,
NANUMBA M. major M. major
M. kieferi M. kieferi 
M. aspericornis
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104
TABLE 3: INFECTIVITY STUDIES WITH ADULT COPEPODS
Copepod Species 
(Adult stages)
Ingested
L,
Maximum Number of L, 
Ingested
Larval Development
(L, ->L3)
YES NO 1 2 3 4+ YES NO
M. aspericornis ✓ s S
M. kieferi v' ■/ S
T. incisus V
T. oblongatus
(Copepodids)
Ingested
L,
Maximum Number o f L, 
Ingested
Larval Development
(L, —>L3)
YES NO 1 2 3 4+ YES NO
M. aspericornis v' y
M. kieferi V
T. incisus s S
T. oblongatus V
(Adult stages) Percentage Ingesting 
“n” Number o f Larvae
Percentage mortality within  
12 hrs of ingesting “n” larvae
1 2 3 4+ 1 2 3 4+
M. aspericornis 2 17 76 5 8 64 82 88
M. kieferi 5 20 68 7 12 70 82 94
T. incisus 44 53 3 0 0 2 2 4
T. oblongatus 40 60 0 0 0 0 0 0
(Copepodids) Percentage ingesting  
“n” Number o f Larvae
Percentage mortality within 
12 hrs o f ingesting “n” larvae
1 2 3 4+ 1 2 3 4+
M. aspericornis 88 12 0 0 0 0 0 0
M. kieferi 91 9 0 0 0 0 0 0
T. incisus 95 5 0 0 0 0 0 0
T. oblongatus 100 0 0 0 0 0 0 0
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105-
FIGURES
FIGURE 1: LIFE CYCLE OF DRACUNCULUSMEDINENSIS
0s in I f f s
___ ______ 1
Juvenile worms ingested 
by copepods and mature 
into infective stage.
Humans are infected when 
they ingest water containing 
infected copepods.
I
Juvenile worms exit the intestinal 
tract and migrate to the 
subcutaneous tissues.
i
Male and female worms mate, 
and the male worms die.
Females produce juveniles, 
blister forms on skin. The 
blister breaks open and 
juveniles liberated into water.
1
Female worms migrate to the 
skin (legs, ankles, or feet 
most often).
(Parasites and Parasitological Resources)
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10&
FIGURE 2: A COPEPOD
SETAE
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DISTRICT MAP OF C.HNANA 1 °  ^
LEGENO
----- International Boundary
----- Regional Boundary
District Boundary
Ca&tel
& Regional Capital
o District Copital
A Field Practice Site
m •>¥<»« Them/itir Mapping [>✓>•,Ion INoTI.CSIR Arcin
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PE
RC
EN
TA
G
E 
O
C
C
U
R
R
EN
C
E
FIGURE 4: MONTHLY DISTRIBUTION OF GUINEA WORM  DISEASE
108
JAN. FEB. MAR. APR. MAY JUN. JUL. AUG. SEPT. OCT. NOV. DEC.
MONTH
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DRACUNCULIASIS
10<?
FIGURE 5: OCCUPATIONAL DISTRIBUTION OF PERSONS WITH
100
I -
j5
%CL
80
60
40LJCO
20
0
LEGEND:
1 = Pupil; 2 = Student; 3 = Farmer; 4 = Fisherman; 
5 = Trader; 6 = Housewife; 7 = Others.
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(10
FIGURE 6: PERCEPTIONS OF THE CAUSES OF GUINEA WORM
111
2  z
Ui
Ul 
C .
3 5 7
CAUSE OF DISEASE
LEGEND:
1 = Walking bare footed; 2 = Wading in pond/dam; J = Witches;
4 = Pond water; 5 = Juju; 6 = God/Natural; 7 = Others; 8 = Don't know
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FIGURE 7: PERCEPTIONS OF THE METHOD OF TRANSMISSION OF GUINEA WORM
7 0 -
oU
oU
111
P 4U
Z
HI□ Qn
UlCLonAj
* »  I1U
A _  _  I I  1U
1 2 3 4 5 6 
METHOD OF TRANSMISSION
1 = Eating with others; 2 = Entry o f  pond by infected person; 
3 = Flies; 4 = Mosquitoes; 5 = Other; 6 = Don '1 know
LEGEND:
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FIGURE 8: PERCEPTIONS ON HOW TO CURE INFECTION
LEGEND:
1 = Ambilhar; 2 = Paracetamol; 3 = Tamale oil;
4 =  Herbs; 5 = Juju-man; 6 = Other; 7 = Don V know
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1 ) 3
FIGURE 9: PERCEPTIONS ON DISEASE PREVENTION
LEGEND:
1 =Filtering all pond water before drinking; 2 Drinking borehole water only; 
3 = Boiling water before drinking; 4 = Regularly using Abate in ponds
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l J4
FIGURE 10: SOURCES OF KNOWLEDGE ON GUINEA WORM  PREVENTION
60
50
Ui 4 0
O
|so
W 2 0
Q.
10
0
3 4
SOURCE
LEGEND:
/  =  Relatives; 2 = Friends; 5 = Health workers; 4 = Teachers; 
5 = Guinea worm Workers; 6 = Pastor/Imam
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PE
R
CE
m
’A
G
E
115"
FIGURE 11: PRINCIPAL SOURCES OF WATER
STANDPIPE BOREHOLE POND/DAM 
SOURCE OF WATER
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PE
R
CE
N
TA
G
E
116
FIGURE 12: ALTERNATIVE SOURCES OF WATER TO PONDS
40
35
30
25
20
15
10
5
0 I t
NONE BOREHOLE RIVER'STREAM RAINWATER PIPEWATER
ALTERNATIVE SOURCE OF WATER
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l i t
FIGURE 13: COMMON ACTIVITIES AT POND SITE
60 
50
LU 4 0
O
2
§ 3 0
u
u J 2 0Cl
10
0
3
ACTIVITY
1 = Swimming; 2 = Bathing; 3 = Washing clothing; 
4 =Urination ; 5 = Wading to fetch water.
LEGEND:
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FIGURES 14 -  21: T/S OF FEMALE DRACUNCIJUJS MEDINENSIS
FIGURE 14: CEPHALIC REGION OF GUINEA WORM
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ll«jl
FIGURE 15: THE PHARYNGEAL REGION
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l i e
FIGURE 16: ENLARGENED VERSIONS OF:
1. The Muscle Cells
2. Hypodennal Cord
3. Cell Nucleus
4. Pseudocoel
NUCLEUS OF A  MUSCLE CELL
HYPODERMAL CORD
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FIGURE 17: MID-ANTERIOR REGION
LARVAE-FILLED
UTERUS
MUSCLE CELLS 
(NON-CONTRACRILE PORTION)
MUSCLE CELLS
(CONTRACRILE PORTION)
CUTICLE
the relatively thicker musculature
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1 j^ -
FIGURE 18: MID-SECTION
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10.3
FIGURE 19: MID-SECTION PRESENTING THE MUSCLATURE
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I ’M
FIGURE 20: LOWER MID-SECTION
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12,5-
FIGURE 21: THE EXTREME POSTERIOR REGION OF GUINEA WORM
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\2&
FIRST STAGE LARVA (L ,)
FIGURE 22:
TAIL
MOUTH
Note the rough cuticle.
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1 2 'f
THIRD STAGE LARVA (L3)
FIGURE 23:
Note the rather smooth cuticle after molting.
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126
FIGURES 24 - 28: CLINICAL PRESENTATIONS OF DRACUNCULIAS1S
FIGURE 24: A GUINEA WORM  ON THE CHEST OF THE PATIENT
LEGEND:
BT = Point o f  blister formation
OL  = Outline o f  Guinea worm  on a patient
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12
FIGURE 25:
GUINEA WORM BEING EXTRACTED FROM  
THE LEFT FOOT OF A PATIENT
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FIGURE 26:
1 3 °
GUINEA WORM BEING EXTRACTED FROM  
THE THORACIC  REGION OF A PATIENT
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FIGURE 27:
GUINEA WORM BEING EXTRACTED FROM  
THE RIGHT HAND OF A PATIENT
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132 -
figure  28:
GUINEA WORM BEING EXTRACTED FROM  
THE PELVIC REGION OF A PATIENT
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1 3 3
FIGURE 29:
Phylogenetic tree for small subunit ribosomal RNA  sequences o f  Dracunculus 
medinensis, D. insignis, and selected Nematoda.
-------------------------------------------P r i a p u l u s  c a u d a t u s
“ “  C h o r d o d e s  m o r g a n i
“ “ “ —  P a r a t r i c h o d o r u s  a n e m o n e s  
T r i c h i n e l l a  s p i r a l i s
Quartets puzzling m aximum  likelihood results are shown using Priapulus caudatus and 
Chordodes morgani as outgroups. Numbers to the left o f  the nodes indicate the quartet 
puzzling support for each internal branch. Scale bar indicates an evolutionary distance o f  0.1 
nucleotides per position in the sequence. Vertical distances are for clarity only. GenBank 
accession numbers o f  the sequences used for analysis: Ascaris suum  AF036587, 
Caenorhabditis elegans L04153, Chordodes morgani AF036639, Dirofilaria immitis 
AF036638, Globoderapallida  AF036592, Haemonchus contortus L04153, Paratrichodorus 
anemones AF036600, Priapulus caudatus X 87984, Strongyloides stercoralis M84229, and 
Trichinella spiralis U60231. The Dracunculus sequences were not yet subm itted to 
GenBank. Both Dracunculus species form  a sister group to a group formed by Ascaris and 
Dirofilaria.
100
S t r o n g y l o i d e s  s t e r c o r a l i s
72
G l o b o d e r a  p a l l i d a
100
100
100
D i r o f i l a r i a  i m m i t i s
96
A s c a r i s  s u u m
H a e m o n c h u s  c o n t o r t u s
100
0.1 C a e n o r h a b d i t i s  e l e g a n s
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1.3 4 -
f i g u r e  30A: The map of the rRNA duster
[Showing the d ifferent parts o f  the genes that codes for ribosomal 
DNA (rDNA)]
Sequenced for speciation Sequenced for typing isolates
IS S rR NA  ITS1 5.8S rRNA ITS2 28S rRNA
SSU-rRNA (or 18S rRNA) -  ITS1- 5.8S rRNA -  ITS2 -  LSU-rRNA (or 28S rRNA).
Figure 30b: D iagram  o f the ribosomal DNA gene.
The regions coding for the 5.8S, 18S, and 28S subunits o f  rRNA are shown by bars; NTS =  
non-transcribed spacer, ETS = external transcribed spacer, ITS = internal transcribed spacer 
regions.
NTS ETS ITS-1 ITS-2
 1— L .  H — [ ~
18S 5.8S 28S
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FI
G
UR
E 
31
: 
MA
P 
OF
 
TH
E 
W
O
R
LD
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136
FIGURES 32 -  34: VERIFICATION OF PCR PRODUCTS
FIGURE 32: AN 1819bp PCR PRODUCT OF THE SSU rRNA (18S) OF  
DRA CUNCUL US MEDINENSIS
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13 7-
FIGURE 33: AN 1165bp PCR PRODUCT OF THE ITS1 rRNA OF  
DRA CUNCUL US MEDINENSIS
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133
FIGURE 34:
RELATIVE SIZES OF AN 1819bp PCR PRODUCT OF THE SSU rRNA (18S) 
AND AN 1165bp PCR PRODUCT OF THE ITS1 rRNA OF DRACUNCULUS 
MEDINENSIS
1819bp PCR PRODUCT OF THE SSL rRNA 
OF DRACUNCULUS MEDINENSIS
1165bp PCR PRODUCT OF THE ITS 1 rRNA 
OF DRACUNCULUS MEDINENIS
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139
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Ward, W ,B„ Belcher, D. W., Wurapa, F. K. and Pappoe M. E. (1978) Perception and 
management o f Guinea worm disease among Ghanaian villages. A framework for 
determining health education planning. Tropical Geographic Medicine. 31: 155-164.
Watts, S. J. (1996). No transm ission o f  dracunculiasis in Egypt for two centuries. World 
Forum. 17(6): 181-183
Watts, S. J. (1986). Human behaviour and the transmission o f  dracunculiasis: A case study 
from the llorin area o f Nigeria. International Journal o f  Epidemiology. Vol. 15. 252-256.
Yelifari, L., Frempong, E., and Olsen, A. (1997) The intermediate hosts o f  Dracunculus 
medinensis in northern region, Ghana. Annals o f Tropical Medicine and Parasitology. 
91(4):403-9.
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APPENDIX A
QUESTIONNAIRE FOR GUINEA WORM DISEASE (DRACUNCULIASIS) SURVEY
DISTRICT: ..............................................................................
SECTION A: BACKGROUND INFORMATION
Ql. Town/Village: ...................................................................
Q2. Sex: l .M a leD  2. Female □
Q3. Age: 1 .0 - 9  □  2. 1 0 -2 9  □  3. 30 -4 9  □  4. 50 -69  □  5. 7 0 + □
Q4. Educational Background:
1. None □  2. Prim./J. S.S. □  3. S..S.S. □  4. College □  5. Polytechnic □  6. University □  
Q5. Occupation:
1. Pupil □  2. Student □  3. Farmer □  4. Fisherman □  5. Trader □  6. Housewife
□
2. 7. Other (specify) ...............................
SECTION B: KNOWLEDGE BASE
Q6. Have you ever suffered from the Guinea worm disease before? 1. Yes □  2. No □
IF YES TO Q6, Go to Q7,
IF NO TO Q6, Go to Q12
Q7. When was this? 1. This year only □  2. Last year only □  3. Last year and this year □
Q8. How many times? 1. Once □  2. Twice □  3. Three times □  4. >Three times □
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Q9. On which part(s) o f  your body did the worm(s) emerge?
1.Hand □  2. Abdomen □  3. Pelvic region/waist □  4. Thoracic region/chest □
5. Thigh □  6. Knee □  7. Foot □  8. Ankle □  9. Other (spec ify ).............................
Q10. During which month(s) did you get the disease?
l.Jan. □  2. Feb. □  3. March. □  4. April □  5. May. □  6. Jun. □
7. Jul. □  8. Aug. □  9. Sept. □  10. Oct. □  11. Nov. □  12. Dec. □
Q11. How long did it take for the worm(s) to completely come out?
1. One - two weeks □  2. Three - four weeks □  3. Two months □  4. > Two months □
IF NO TO Q6,
Q12. Have you ever heard o f  the disease before? 1. Yes □  2. No □
Q13. I f  yes to the above question, indicate source (i.e, from whom did you hear this)?
1. Mother □  2. Father □  3. Friend(s) □  4. O ther family members □  5. Teacher(s) □
6. Guinea Worm Workers □  7. Pastor □  8. Doctor/Nurse □  9. Other (spec ify )......
Q14. Has any member o f  your family had a Guinea worm disease before? 1. Yes □  2. No □  
Q15. I f  yes to the above question, When w;as this?
1. This year □  2. Last year □  3. Last year and this year □
Q16. What do you think causes the guinea worm disease?
1. Walking bare footed □  2. Wading in pond/dam/stream □  3. Witches □
4. Drinking water from a pond/dam/stream □  5. Juju □  6. It is naturally in our bodies □  7. 
Other (specify)..................................
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Q17. A person who has Guinea worm may spread the disease by?
1. Eating with others □  2. Washing the sore in a pond/stream/dam □  3. Flies □
4. Mosquitoes □  5. O ther (spec ify )........................................
Q18. Can guinea worm disease be cured? 1. Yes □  2. No □
Q19. If yes to question 18, how can the disease be cured? By using
1. Ambilhar □  2. Paracetamol □  3. Tamale oil □  4. Herbs 5. A Jujuman □
6. Other (specify)........................................................................................
Q20. If yes to question 18, How did you learn about this cure(s)?
1. Mother □  2. Father □  3. Friend(s) □  4. Other family members □  5. Teacher(s) □
6. Guinea Worm Workers □  7. Pastor □  8. Doctor/Nurse □  9. Other (specify )......
Q21. Can the disease be prevented? 1. Yes □  2. No □
Q22. If yes to question 21, effective and affordable methods includes:-
1. Filtering all pond/stream/dam water before drinking □
2. Drinking Bore hole water □
3. By boiling water before drinking □
4. Abating ponds/dams □
Q23. How did you learn about this preventive measure(s)?
1. Mother □  2. Father □  3. Friend(s) □  4. O ther family members □  5. Teacher(s) □
6. Guinea Worm Workers □  7. Pastor □  8. Doctor/Nurse □  9. Other (spec ify )...
Q24. If no to question 18, W HY ?...............................
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SECTION C: HUMAN PRACTICES
Q25. Where do you get your drinking water from?
1. Stand pipe □  2. Bore hole □  3. A pond/dam/stream □  4. Other (spec ify )....................
Q26. If pond/dam is not mentioned in Q25 above, do you use pond/dam water during any part 
of the year? I . Yes □  2. No □
Q27. If pond/dam mentioned in Q25 above, how much do you depend on the pond water? 1.1. 
All year round □  2. Only in the dry season □  3. Only in the rainy season □
Q28. Which other source(s) o f  water do you use?
1. None □  2. Bore hole □  3. River/stream □  4. Rain water □  5. Pipe water □
Q29. Which o f  the following do you do in the water?
1. Swimming □  2. Bathing □  3. Washing clothes □  4. Urinating □
5. Wading through to fetch water for domestic/farm use.
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APPENDIX B 
WORM MORPHOMETRY
153
j WORM WEIGHT
( g )
) WORM LENGTH WORM 
j DIAMETER (mm)(cm)
1.217 62.7 1.35
0.677 37 1 .2
0.397 45.2 0.75
1.1 65.1 1.23
1.049 53 1.35
0.549 37 0.88
0.437 37 1.15
1 .8 77.8 1.961
1.55 69 1.425
1.675 55.2 1.44
0.831 63 .4 1.25
1.159 70.8 1.475
1.246 73.3 1.375
1.021 50.6 1.65
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APPENDIX C 
DNA EXTRACTION FROM ANIMAL TISSUE  
Bio 101 FastDNA Kit 
a) PULVERIZING OF TISSUE AND LYSING OF CELLS
1. Wash tissue sample thoroughly in cold PBS to remove 70% EtOH..
2. Cut about 30-50mg (approximately 2-3cm) o f  worm and rehydrate in 1,000|il CLS-TC 
buffer for 18-24hrs at room temperature. Spin 1 minute at 10,000xg. Pipette off Buffer.
3. Add 500(_il o f  CLS-TC buffer and homogenize with an electric homogenizer in a tube.
4. Add the resulting pulp o f  tissue to lysing matrix 4 (1/4" sphere + garnet + 1/4" cylinder
5. Add 850|il o f  CLS-TC buffer to the tube. Must leave a minimum air space o f250 |il to allow 
for grinding action in FastPrep.
6. FastPrep at setting 5 for 20 seconds.
7. Spin 10 min at 10.000-x g (Jouan A -14 @ 10.700 rpm).
8. If intact tissue is seen, FastPrep again at setting 5 for 20 sec and spin again.
b) BINDING OF DNA TO MATRIX, WASHING AND ELUTION
9. Spin again in M icro-centrifuge for 5minutes at 14000-Xg to pellet protein and cell debris.
10. Transfer 600[il o f  supernatant to a clean tube containing 600ul o f  binding matrix.
11. Mix gently and incubate at room temperature for 5 min.
12. Spin 1 minute and discard supernatant.
13. Gently re-suspend pellet in 500(.il o f  SEWS-M to wash DNA.
14. Transfer suspension to SPIN filter.
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15. Spin 1 minute and discard contents o f  catch tube.
16. Spin again 1 minute to dry.
17. Transfer SPIN filter to a clean tube and add 100^1 o f  DES to elute DNA.
18. Gently vortex to re-suspend and incubate at room temperature for 3 minutes.
19. Spin 1 minute.
20. DNA is in the flow through.
21. It may be necessary to perform QIAquick purification to remove PCR inhibitors. Use 5ul 
purified DNA/50ul PCR Reaction.
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APPENDIX D 
QIAquick-Spin: PCR Purification Kit
This protocol is designed for purification o f  single or double-stranded PCR products from 
primers, nucleotides and polymerases.
NOTE:
• Add 220ml of ethanol (96-100%) to buffer PE before use.
• This protocol calls for the u seo f2m l micro-centrifuge tubes, which are included 
in the kit.
• The entire procedure is conducted at room temperature.
• All spins are top speed for a conventional, tabletop micro-centrifuge. 
PROCEDURE:
1. Add Buffer PB (this should be 5 times the Volume o f  DNA), to the sample (extracted 
Genomic DNA). Thus, if  Extracted DNA is 100|il. then add 500|il o f Buffer PB. Mix 
thoroughly with micropipette.
2. Apply the above mixture to the supplied QIAquick-spin column (contained in a 2ml micro­
centrifuge tube).
3. Centrifuge for 60 seconds at maximum speed.
4. Discard flow-through, from the catch-tube. Blot tube on tissue paper. Do not discard the 2ml 
micro-centrifuge tube. Insert the spin column back into the just used 2ml tube.
5. Wash QIAquick-spin column by adding 750|.il o f  Buffer PE. Centrifuge for 60 seconds.
6. Discard buffer PE flow-through from the tube. Blot tube and insert spin column back into the 
tube.
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7. Spin for an additional 60 seconds to get rid o f  excess buffer PE.
8. Now remove and place Q1 Aquick-spin column in a clean (labeled), 1,5ml micro-centrifuge 
tube.
9. Elute DNA by adding 50|il o f  BE (10 mM Tris/HCl, pH = 8.5) or TE. Waite 2 minutes, and 
Centrifuge for 60 seconds.
10. DNA is in the flow-through.
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APPENDIX E
StrataPrep PCR PURIFICATION
1. Add an equal volume o f  DNA binding solution to PCR product in a siliconized tube. Mix
with the pipette. (Do not go above 350p.l o f  each).
2. Transfer above solution/mixture to a micro-spin cup in a 2ml catch tube.
3 . Spin at maximum speed for 30 seconds.
4. Discard binding solution, keep the micro-spin cup and catch tube.
5. Add 750(0.1 wash buffer to the m icro-spin cup and spin 30 seconds.
6. Discard wash buffer, keep the micro-spin cup and catch tube.
7. Spin the micro-spin cup in the catch tube again to remove residual alcohol.
8. Transfer the micro-spin cup to a siliconized tube. Discard tube.
9. Add 50)0.1 de-ionized UV H20  onto the fiber matrix at bottom o f micro-spin cup.
10. Incubate 5minutes at RT.
11. Spin30 seconds.
12. Transfer the contents to a labeled siliconized tube.
13. For use in cycle sequencing, run a gel again to determine the amount o f template 
(volume) to use in the sequencing reaction. This you determine by the intensity o f the bands 
(i.e., 3, 5(il).
NB: The weaker the band, the more templates you need to use.
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APPENDIX F 
CENTRI-SEP PROTOCOL
(FOR PURIFYING PCR PRODUCTS FOR SEQUENCING REACTIONS)
Column Hydration
Gently tap the column to ensure that the dry gel has settled in the bottom o f the spin column.
1. Remove the top column and reconstitute the column by adding 0.80ml o f  reagent grade water 
to buffer. Leave the column end stopper in place so column can stand up by itself. Replace 
the column cap and hydrate the gel by shaking and inverting the column or vortexing briefly.
It is important to hydrate all o f  the dry gel.
2. Allow at least 30 minutes o f  room temperature hydration time before using the columns.
Removal o f Interstitial Fluid
3. Remove air bubbles from the column gel by inverting the column and sharply tapping the 
column, allowing the gel to slurry to the opposite end o f  the column. Stand the column up 
and allow the gel to settle while in a microtube rack.
4. After the gel has settled and free o f  bubbles, first remove the top column cap. and then the 
bottom end stopper from the bottom.
5. Allow excess column fluid to drain (gravity) into a wash tube (2ml). If  the fluid does not 
begin to flow immediately through the end o f  the column, use a 2ml latex pipette bulb to 
apply gentle air pressure to the top o f  the column filter. The column wall stop draining on its 
own. Approximately 200-250j.il will drain from the column. Discard this fluid.
6. Spin the column and wash tube in a variable speed centrifuge at 750Xg for 2 minutes to
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remove interstitial fluid. For an Eppendorf microcentrifuge Model 5415C, spin at 3,000 rpm.
7. Approximately 300j.il o f  fluid will be removed. If  there is a drop at the end o f the column, 
blot it dry. Discard the wash tube and the interstitial fluid. Do not allow the gel to dry 
excessively. Process the sample within the next few minutes.
Sample Processing
8. Hold the column upright and transfer 20)_il o f  completed DyeDeoxy terminator reaction 
mixture to the top o f  the gel. Carefully dispense the sample directly onto the center o f the gel 
bed at the top o f  the column, without disturbing the gel surface. Do not contact the sides of 
the column with the reaction mixture or the sample pipette tip, since this can reduce the 
efficiency o f  purification and possibly ruin the analysis due to excess dyes.
9. Place the column in the sample collection tube (1.5ml) and place both into the rotor. 
Maintain proper column orientation. The highest point o f  the gel media in the column 
should always point toward the outside o f  the rotor. Spin the column and collection tube at 
3,000 rpm for 2minutes. The purified sample will collect in the bottom o f the Sample 
Collection Tube. Discard the spin column and proceed with the Sequencing sample 
preparation procedure. Protect sample from light with an aluminium foil.
10. Dry the sample in a vacuum centrifuge. Do not apply heat.
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APPENDIX G 
BASE SEQUENCES OF ITS1 OF RIBOSOMAL DNA OF DRACUNCULUS ISOLATES
1. SPECIMEN OF HUMAN ORIGIN FROM BURKINA FASO
AACATGGTCGCATGCAATAACGCACCCTACTACACTGGGGACTCAACGTGCTATGTC
CATTGTCGAAAGGCATTGGTAACCCGTTGAAAATCCTCCGTGCTCGGGATAGGGAAT
TGCAATTATTTCCCTTGAACGAGGAATCCCTAGTAAGTGTGAGTCATCAGCTCACGC
TGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTGCCCGGGACTGAGCCGTT
TCGAG AAAAGCGGAGACTGCTGT ATTGAAGCCGAAT ATTT GCGGTGGAAAT ACTCT
GGTGGAAATCGCCTTAATCGCAGTGGCTTGAACCGGGCAAAAGTCGTAACAAGGTT
TCCGTAGGTGAACCTGCGGAAGGATCATTAACGTATTTGGAAAGCATATTAAAATAC
GCG AT ATG AT A ACG AT AGTT GT ATCG A A ATT GGTTT G AAG ATTTTT CG AT AAAG ACG
AGAACG ACG AATCT AT AT ATCGCGAC AATT AT GT AT A A ACGT A ATTTT AT AAAAAAT
AATGCGTTTGATCAAAAGAATGATGCATGGGTCGTCTTCGCGTCGTCATCATMGTAT
GTCGTTGT AATG ATGATG ATG AT G ATGAT GAT GT AGT ACTT AT GC ATG AC GGTT AAT
GAATAACGCAT ATTTTGC AT AT AC ATAT ATTGCGTG AT ATT ATT GTT GTT AT AT GAT G
AT A ACTCGTT A ATTTCGT G A A AG AATTTT C AAT AT AT ATTT CG AT AC ACTCG AA A A A
AATGTACAGAATAAAGAAGTAAAATGCGTGATTTTGTACAAATAACAGTGACACGG
TTGGCGTCTATACGTTGTTTAGTAGTTATTGCCCGACTGTCAGTAACGTTTGAACGAC
GGCGATATAGTTCTCGATGTGAGAGGAAATTTTCAAATTCGAGAATAGACTTAATAA
GTATTGCAGGGATACTGCCAACAAGAAAAAAATTCAATAAAGAAATTCATCTTAATT
AAGAATATGATAAAACGTTTCAAATAATGATATGTATATATTTTTGAAATGGATTGA
TGGGAGATAATGGCCCGAAAAATGAACTGTAGTATATCTTCATTTTCGTTATTATTAT
CACCC ATC ATC ATC AA AT AT AT AT AC AATT ATT ATT GT AT A AG AAGCC AAAAG AT GA
GACATTCTTAACAGA
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2. SPECIMEN OF CANINE /DOG  OR IG IN  FROM  GHNANA
AACATGGTCGCATGCAATAACGCACCCTACTACACTGGGGACTCAACGTGCTATGTC
CATTGTCGAAAGGCATTGGTAACCCGTTGAAAATCCTCCGTGCTCGGGATAGGGAAT
TGCAATTATTTCCCTTGAACGAGGAATCCCTAGTAAGTGTGAGTCATCAGCTCACGC
TGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTGCCCGGGACTGAGCCGTT
TCGAGAAAAGCGGAGACTGCTGTATTGAAGCCGAATATTTGCGGTGGAAATACTCT
GGTGGAAATCGCCTTAATCGCAGTGGCTTGAACCGGGCAAAAGTCGTAACAAGGTT
T C C G T AG G TG  A  ACCTGCGG AAGG ATC ATT A ACGT ATTTGG A A AGC AT ATT AAAAT AC
GCGATATGATAACGATAGTTGTATCGAAATTGGTTTGAAGATTTTTCGATAAAGACG
AGAACGACGAATCTATATATCGCGACAATTATGTATAAACGTAATTTTATAAAAAAT
AATGCGTTTGATCAAAAGAATGATGCATGGGTCGTCTTCGCGTCGTCATCATMGTAT
GTCGTTGTAATGATGATG ATG ATG ATG ATG ATGT AGTACTTATGC ATG ACGGTT A AT
GAATAACGCAT ATTTTGC ATAT AC AT AT ATTGCGT GAT ATT ATTGTT GTTATAT G ATG
ATAACTCGTTAATTTCGTGAAAGAATTTTCAATATATATTTCGATACACTCGAAAAA
AATGTACAGAATAAAGAAGTAAAATGCGTGATTTTGTACAAATAACAGTGACACGG
TTGGCGTCTATACGTTGTTTAGTAGTTATTGCCCGACTGTCAGTAACGTTTGAACGAC
GGCGATATAGTTCTCGATGTGAGAGGAAATTTTCAAATTCGAGAATAGACTTAATAA
GTATTGCAGGGATACTGCCAACAAGAAAAAAATTCAATAAAGAAATTCATCTTAATT
AAGAAT ATG AT AAAACGTTTC A A AT A ATGAT AT GT ATAT ATTTTT G AAATGG ATTG A
TGGGAGAT AATGGCCCG AAA A AT G AACT GT AGT AT ATCTTC ATTTT CGTT ATT ATT AT
CACCCATCATCATCAAATATATATACAATTATTATTGTATAAGAAGCCAAAAGATGA
GACATTCTTAACAGA
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3. SPECIMEN OF HUMAN ORIGIN FROM PAKISTAN
a a c a t g g t c g c a t g c a a t a a c g c a c c c t a c t a c a c t g g g g a c t c a a c g t g c t a t g t c
CATTGTCGAAAGGCATTGGTAACCCGTTGAAAATCCTCCGTGCTCGGGATAGGGAAT 
TGCAATTATTTCCCTTGAACGAGGAATCCCTAGTAAGTGTGAGTCATCAGCTCACGC 
TGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTGCCCGGGACTGAGCCGTT 
TCG AG A A A AGCGG AG ACTGCT GT ATT G A AGCCG AAT ATTT GCGGT GG AAAT ACTCT 
GGTGGAAATCGCCTTAATCGCAGTGGCTTGAACCGGGCAAAAGTCGTAACAAGGTT 
TCCGT AGGTG A ACCTGCGG A AGG ATC ATT A ACGT ATTT GG AA AGC AT ATT AAAAT AC 
GCGATATGATAACGATAGTTGTATCGAAATTGGTTTGAAGATTTTTCGATAAAGACG 
AG AACG ACG A ATCT AT AT ATCGCG AC A ATT AT GT AT AAACGT AATTTT AT AAAAAAT 
AATGCGTTTGATCAAAAGAATGATGCATGGGTCGTCTTCGCGTCGTCATCATMGTAT 
GTCGTTGT AATG AT GAT G ATG ATG ATG AT GAT GT AGT ACTT AT GC AT G ACGGTT AAT 
G AAT A ACGC AT ATTTT GC AT AT AC AT AT ATT GCGT GAT ATT ATT GTT GTT AT ATG AT G 
ATAACTCGTTAATTTCGTGAAAGAATTTTCAATATATATTTCGATACACTCGAAAAA 
A ATGT AC AG A ATA AAG AAGT A A AATGCGT G ATTTT GT AC AAAT AAC AGTG AC ACGG 
TTGGCGTCT AT ACGTT GTTT AGT AGTT ATT GCCCG ACT GTC AGT AACGTTT G AACG AC 
GGCG AT ATAGTTCTCG AT GTG AG AGG A A ATTTTC AA ATTCG AG AAT AG ACTT AAT AA 
GTATTGC AGGGAT ACTGCC AAC AAG AAA A AAATTC AAT AAAG AAATTC ATCTTAATT 
AAG AAT ATG AT AAA ACGTTTC A A AT A AT GAT AT GT AT AT ATTTTT G AA AT GG ATT G A 
TGGG AG AT A ATGGCCCG AA A A ATG A ACTGT AGT AT ATCTTC ATTTT C GTT ATT ATT AT 
CACCC ATC ATC ATC AAAT ATATATAC AATT ATT ATT GT AT AAG AAGCC AAAAG AT G A 
GACATTCTTAACAGA
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