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This is to certify that the

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Immune Responses in Onchocerciasis
Correlate with Microfilarial Destruction
and Treatment History

presented by

Magdi Mahmoud M. All

has been accepted towards fulfillment
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Master degree in Microbiology

and Molecular Genetics

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Charles D. ackenzie

 

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6/01 C'JCIRCJDaiODUOpGS-DJ 5

Immune Responses in Onchocerciasis Correlate
with Microfilarial Destruction and Treatment
History

By

Magdi Mahmoud Mohamed Ali

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

'MASTER OF SCIENCE

Department of Microbiology and Molecular Genetics

2002

ABSTRACT

Immune Responses in Onchocerciasis Correlate with
Microfilarial Destruction and Treatment History

By
Magdi M. Ali

Onchocerciasis, or river blindness, is a parasitic disease that affects
more than 20 million people in the world. It presents clinically with a wide
spectrum of dermal and ocular manifestations, the basis of which is
believed to involve the immune system. The induction of pathology is
directly related to the presence of the microfilarial stages of this filarial
nematode. The aim of this study is to gain better understanding of the role
of immune response in the host reactions to Onchocerca volvulus
parasite. Patients with either of two major forms of the clinical spectrum-
mild/asymptomatic (n=12) and severe dermatopathology (n=16) were
studied by assaying the antigen-driven proliferation of peripheral blood
mononuclear cells and the ability of patients’ serum antibodies to promote
cytoadherence activity to microfilariae in vitro. Immune responses of those
with severe disease were found to be stronger compared with the mild
dermatopathology group. Mectizan® treatment was followed by an
increase in immune responsiveness in those with initially poor responses.
Thus the degree of dermatopathology is related to the host’s immune

response against microfilariae and immunocompetence may be necessary

for Mectizan® to clear the infection efficiently.

To my loving wife Dr. Suzan
and

to my children

iii

Acknowledgments

This work could not have been completed without the continuous support,
guidance and encouragement of my mentor and sincere friend Prof.
Charles Mackenzie. l have learned much from his advice, patience and
foresight through out the study period and the long-term research
collaboration.

I owe special thanks to Prof. Mamoun Homeida at the Academy of
Medical Science and Technology, Khartoum, Sudan for his support and
advice.

My gratitude extends to Dr. Omer Z. Baraka at the Medical School,
University of Khartoum, Sudan for the clinical classification for some of the
patients involved in this study. My gratitude also goes to Prof. Suad
Suleiman, Director, Tropical Medicine Research Institute, Khartoum,
Sudan.

My utmost appreciation is extended to Dr. Ron Patterson, Dr. Jeffery
Williams and Dr. Walt Esselman for their valuable advice, encouragement
and support.

Special thanks and deepest gratitude are reserved for my family: My
parents who, lack formal higher education themselves, have always
encouraged my pursuits, and my dear wife Dr. Suzan who besides being a
treasure for continuous support, has also carried out the clinical
classifications for some of the patients included in this study.

Thanks to them all.

iv

List of contents

Listoftables

List of figures

ListofAbbreviations

Chapter one

Generalintroduction .. ...
Clinical manifestations............................................................
Immunology and immunopathology.......................................
Diagnosis
Treatment
Rationale and objectives

References

Chapter two

Immune Responses against microfilariae are associated

with the severity of onchodermatitis
Introduction
Patients and Methods.....................
Results
Discussion

References ..

vii

viii

16

22

25

3O

32

48

.49

51

56

58

.62

Chapter three

lmmunocompetence may be important in the effectiveness

of MectizanR (lvermectin) in the treatment of human

onchocerciasis...... .........68
References 74

Summary 78

vi

Table1
Table2

Table 3

List of Tables

vii

.65

.66

75

FigZ

List of Figures

viii

...67

..76

..77

.79

List of Abbreviations

Mf, microfilaria, OV, Onchocerca volvulus; L3, third Larval stage;
pers.comm., personal communication; WHO, World health Organization;
TH1/2, T helper 1, Th2; IL, Interleukin; TCR, T-oell receptor,lgE,
Immunoglobulin E; PHA, Phytohemagglutinin; OVA, Onchocerca volvulus
antigen; RPMI, Rosewell Park Memorial Institute; PBMC, peripheral Blood
Mononuclear cells; HFCS, heat-inactivated fetal calf serum; cpm, count
per minute; SI, stimulation index; CMI, cell-mediated immunity; AMI,

Antibody-mediated immunity; OD, optical density.

ix

Chapter 1.

Introduction

General Introduction

F ilariae are a distinct group of nematode parasites. All those that infect
humans are classified in the family Onchocercidae and the superfamily
Filariodea. The name filaria is derived from the Latin word “filum” meaning
thread, and refers to the thread-like morphology of the adult worm. Filariae share
common features, including an adult stage that resides outside the digestive
tract, the requirement of an obligate hematophagus arthropod vector as an
intermediate host, and the release of motile offspring called microfilariae into the
vertebrate host. F ilarial worms continue to present a serious challenge to those
concerned with public health and to control programs in many parts of the world.
Seven principle species of filariae infect humans, and these are typically grouped
into three categories. The lymphatic filariae, consisting of Wucheren'a bancrofti
and Brugia malayi, reside as adults in the lymphatics and infect some 120 million
persons (Ottesen et al, 1997). Infection with these parasites may cause acute or
chronic obstruction of the lymphatics that ultimately leads to elephantiasis.
Onchocerciasis is caused by Onchocerca volvulus; the adults live in
subcutaneous nodules or in deeper skeletal tissues associated with major bones.
The remaining filariae including Loa Ioa, Mansonella perstans, Mansonella
ozzardi, and Mansonella streptocerca, are less important as causes of disease
and suffering, although L. Ioa can provoke temporary inflammatory swelling

(calabar), hypereosinophilia and other allergic reactions. Loa loa’s habit of

migrating widely throughout the body is especially disturbing if it crosses the eye
and surrounding tissues.

Adult filariae are typically long, slender worms, with pronounced sexual
dimorphism; females are characteristically much longer than males. Adult worms
are observed rarely because of their location in the host, so the microfilariae are
the usual diagnostic stage. Microfilariae are relatively undifferentiated when
released by female worms. In some genera of filariae (e.g., Wuchereria, Brugia,
and Loa), the microfilariae retain the egg membrane as a sheath, whereas the
others release unsheathed microfilariae (e.g. Onchocerca). Once released by the
female worms, microfilariae make their way into the circulation, or, in the case of
O. volvulus and M. streptocerca, into the skin. They are generally not infective for
non-human vertebrate hosts, undergo no further development in the humans,
and survive for 1-2 years. Microfilarial periodicity is a phenomenon shown by
some filarial species and it can pose a difficulty for their parasitological diagnosis
(Mason-Bar and Aped, 1982).

Onchocerciasis or ‘river blindness’, one of the major filarial diseases of
humans, is caused by O. volvulus, and transmitted by female black flies of the
genus Simulium. Onchocerciasis is a leading cause of preventable blindness and
a severe pruritic skin condition in endemic areas, and thus one of the most
important public health and socio-economic problems faced by the rural
populations in these areas (WHO, 1976). It is estimated that over 90 million
people are at risk of infection, with more than 20 million being infected. The

majority of patients suffer from variable skin lesions. One million people suffer

visual impairment as a result of onchocerciasis, with at least 340,000 cases of
blindness attributable to the disease. More than 46,000 people lose their vision
every year as a result of this devastating disease (WHO, 1995). The impact of
blindness on a community is reflected in an increased mortality rate: the mortality
amongst blind people is four times higher than that of non-blind persons of the
same age in a community (WHO, 1994).

Onchocerciasis is endemic in large areas of Africa, on the Arabian
Peninsula, as well as in central and South America. It mainly affects small
isolated and more remote communities, with the result that some endemic areas
have escaped detection and many thousands of villagers with onchocercal eye
disease probably remained undiagnosed. In general, a favorable ecology forthe
intermediate host black fly determines the distribution of the disease. The
disease is usually prevalent in rural areas around water sources where the vector
prevails. The vector breeds in fast flowing rivers, hence the name “river
blindness”. The effect of the disease in communities includes the deterioration of
living conditions and desertion of fertile riverside lands by the villagers (WHO,
1987). Fear of the disease has led to the depopulation of river valleys where the
vector breeds. Therefore, the disease poses a significant obstacle to
socioeconomic development. There are over 700 species of Simulium around the
world. The black fly 8 .damnosum is the main vector of O. volvulus in Africa
(Crosskey, 1969). Several subspecies of S. damnosum are distributed
worldwide. The male black flies feed only on plant juices and have no role in the

transmission of onchocerciasis.

The female flies feed on blood and can take up to 1 mg of blood at each
meal. The eggs are laid 3-5 days after the meal. Within 24 hours of laying the
eggs, the female fly takes another blood meal. The bite, which involves the
creation of a blood pool under the epidermis, is not always noticed immediately,
but may be followed by debilitating severe itching. Simulium can fly for up to 80
km in 24 hours along the water sources by the aid of their strong thoracic
muscles. They may even go hundreds of kilometers with the help of wind from

one river basin to another.

Life Cycle of Onchocerca volvulus:

The life cycle of the parasite in the host still holds some mysteries, due to
the lack of a truly suitable comparable animal model and the dearth of
postmortem information. The life cycle stages take place in two hosts, the human
primary host and the black fly as the intermediate host. As an infected female
black fly takes a blood meal from the human, it injects the infective parasitic
larvae (known as L3) into the skin. It is proposed that the larvae penetrate the
superficial layers of the skin, migrate and develop into adult worms elsewhere in
the body over a period of 6-12 months. Within 1-3 years after infection,
onchocercal nodules appear in subcutaneous tissues, usually at sites of bony
prominences such as the hips and ribs. These nodules are formed around coiled
mature worms and are encapsulated by host reactive tissues with a rim of
vascularized fibrous tissue; they usually contain both male and female adult

worms. The adult males are 2 —5 cm in length and 0.02 mm in diameter, while

the females are 50 —70 cm in length and 0.04-0.06 mm in diameter. Fertilized
females give birth to live embryos, about 500,000 to 1,000,000 per year through
out her sexually active life of 8-12 years. It has been estimated that the total
number of microfilariae distributed throughout the dermis, the eyes, and other
parts of the body, in heavily infected individuals may reach 200 million. They are
not commonly found in blood, but may be detected in the urine. The microfilariae
may live for > 2 years

0. volvulus develops through a number of stages in the female black fly.
On biting an individual with dermal microfilariae, the fly ingests microfilariae,
which pass to the stomach, where most of them are digested. However a few
may survive and then pass to the thoracic muscle, where they moult twice to
become infective filariform larvae measuring 650 microns in length. These latter
forms migrate to the tip of the proboscis in the mouthparts and are transmitted to
a human during a subsequent blood meal. The number of infective larvae in one
black fly is usually < 10 (WHO, 1987). The cycle of development in the black fly
takes about 7 days at 27 to 30 °C, but can last up to 10 or 12 days at lower
temperatures. Development stops when temperature falls below 16° C, as often
happens in the night (Duke, 1968). O.volvulus can also be pathogenic to the
black fly: on biting an individual with very heavy loads of microfilariae, the fly may
ingest several hundred microfilariae which can destroy the fly’s own tissues
during migration and development, thus preventing flight and other functions

(WHO, 1987).

F ig.1 Life Cycle of the parasite

Oncho cerca volvulus

/

Subcutaneous tissue

Enters skin through fly bite wound

  

HOMO SAPIENS

 

 

‘ lnfective stage

Migrates to head and proboscis

SIMULIUM Penet'rates storflach wall
Thoracic muscles
/

3rd stage larva

Larva (sausage form)
\ 6% <’

Clinical Manifestations:

The pathogenesis of filarial diseases is characterized by acute and chronic
inflammation. The parasite, the immune response, and/ or consequent
opportunistic infections initiate these inflammatory responses.

The clinical changes seen in human onchocerciasis are believed to be
associated with the destruction of the microfilarial stage of O. volvulus in skin and
eyes. Clinical disease is caused by the microfilarial stages of O.volvulus, with
inflammation and tissue damage mostly seen when the parasite is dying or being
destroyed by drug and/or host responses. A papular eruption in the skin leads to
varying degrees of dermal and epidermal pathology that is complicated by self-
inflicted trauma due to the intense pruritic nature of the infection. Thus, the
consequent pathology represents the cumulative tissue and functional outcomes
of a long-standing interplay between host and parasite, often lasting many
decades. Severe skin changes take place making it one of the most devastating
infectious diseases of human skin. However, the disease is best known for its
ocular pathology, which can affect many parts of the eye causing irreversible
damage and blindness. The adult male and female worms are encapsulated in
subcutaneous nodules, usually palpable on bony prominences such as iliac
crest, hip, ribs knees and skull.

The period between infection with infective larvae and the production of
microfilariae by fertilized adult worms (the prepatent period) varies from 7 month
to more than 2 years. Generally, clinical symptoms are not present during this

period. However, the immature worms may stimulate some immunological

responses (Duke, et al 1991 ). The time from invasion by infective larvae to the
development of clinical signs (the incubation period) is usually longer than the
prepatent period and may last for many years; it is much longer for the

manifestation of the ocular disease than the dermal presentations (WHO, 1987).

Dermal Onchocerciasis

A broad spectrum of skin manifestations can result from inflammatory
reactions associated with damage due to the disintegration of the microfilariae.
During the early skin lesions, mf are found primarily in the dermis, and cause no
observable reaction as long as they are alive. It is only after the death of mf that
inflammatory responses appear. However, it is debatable as to which comes first,
parasite death or the host’s responses. Circulating antigens released from
disintegrating mf may be deposited into blood vessels and connective tissue in
the skin and result in pathology. The clinical manifestations vary according to the
microfilarial load in the skin, the immune responses of the human host, and the
duration of the infection (Mackenzie et al 1987; Murdoch, 1992). Eosinophils,
neutrophils and macrophages aggregate around dead or dying microfilariae. The
commonest early symptoms of the disease include irritation of the skin, pruritus
that can be accompanied by oedema and a sensation of hypothermia. During the
early stages, the signs are often confined to one part of the body. Chronic
infection may present as depigmentation (leopard skin), a striking feature seen
mostly in the shins and has been used for rapid assessment of the prevalence of

the disease (Edungbola et al 1987). These lesions are white and interrupted by

foci of persistent pigmentation around the pores and hair follicles. The areas may
be slightly depressed or atrophic. In later stages, the skin loses elasticity and
may become wrinkled and hang in loose folds (hanging groin) (Salih, 1985); this
is often associated with local underlying lymphadenopathy (Mackenzie,
pers.comm.).

Dermal onchocerciasis has two forms: a “generalized form” which is more
prevalent with high microfilarial load, and a “localized form" manifesting as a
chronic hyper-reactive, onchodermatitis or sowda. Sowda is the Arabic word for
black, and the condition was first described in Yemen (Fawdry, 1957) and then in
West Africa (Bartlett et al, 1979) and in eastern Sudan (Ghalib et al, 1987). The
affected area of skin is intensely pruritic, dark and thickened. There is an
extensive inflammatory cell infiltration (plasma cell, eosinophils, occasional
lymphocytes and histiocytes) that forms broad cuffs around dermal vessels,
greatly thickens the dermis, and causes sclerosis, edema, hyperpigmentation
and papular eruption. Moreover, the regional lymph nodes are soft and greatly
enlarged but usually not tender. The condition is usually localized and typically
involves only one lower extremity. The live microfilarial load is extremely low and
even sometimes undetectable. Severe pruritic skin rashes with acute
inflammatory reactions may also occur in individuals who are visitors and not
from the endemic area when they became infected for the first time; they are
immunologically hyper-responsive to the parasite and its antigens. This could be
due to strain differences in the parasite or HLA differences in the patients. HLA-

00810201 has been found to be associated with sowda (Danfack et a/ 1999).

10

Cooper et al (2001) confirmed the notion that early exposure and early parasite
patency are associated with a vigorous cellular response, unlike chronic
infections in which the cellular response becomes down regulated partly through
an lL-10— dependent mechanism. Moreover, host differences in attractiveness for
the vector might also affect the immunity to this disease. However Kruppa &
Burchard (1999) showed that infected flies similarly bite infected and non-
lnfected (putatively immune individuals).

A new candidate for the cause of the tissue reactions in filarial infections
are the Wolbachia organisms carried by this parasite, which are proposed to be
major stimulators of inflammation (Taylor et al 2000). The impact of pruritus is
definitely reflected on the general health appearance of the infected individuals
leading to noticeable weight loss in people in the hyperendemic areas (Burnham,

1991).

Nodules (Onchocercomata)

Many patients develop fibrous subcutaneous masses containing the adult
onchocercal worms know as “nodules”. These nodules are painless in
themselves (unless pressing on a vital organ), mobile and 0.5 -1.0 cm in
diameter. They can group together to form conglomerates as big as 6-8 cm in
diameter containing 10-15 individual worm masses. They are mostly seen over
bony prominences of the trunk, thigh, shoulders, arms and cranium (Krupp 8.
Chatton 1978). However, some nodules lie deeper and are impalpable. The sites

of the nodules depend in part on the biting habits of the vector black fly (Duke,

ll

1972). For example it is mostly seen in South America on the skull due to this
biting habits.

The composition of the nodules encasing adult onchocercal worms
includes an outer capsule of fibrous tissue, an inner dense inflammatory cell
infiltrate surrounding the enclosed adult worm and a less dense layer of chronic
inflammatory cells between the outer and inner layers. The character of the
infiltrate varies in terms of the presence or absence of eosinophils but
macrophages are almost always the predominant cell type. Lymphocytes are
most abundant at the periphery of the nodule and surrounding the inner core of a
dense inflammatory cell infiltration composed primarily of macrophages around
the adult worm itself. lmmunohistological and cytochemical staining of the
various cell population in the nodules show that infiltrating macrophages closest
to the worm stain strongly positive for complement receptor CR4 (CDl1, CD18)
and for MRP8/MRP14 (an intracellular calcium-binding protein involved in
chemotaxis and inactivation of certain cells), while they are variably positive for
CD68 (a marker for dendritic cells), Fc receptor FcR1 (CD64), and HLA class II
(activation marker) (Gatrill eta/1987; Edgeworth eta/1993). Interestingly,
macrophages seem to be the cell type most intimately involved with adult
parasites, while the eosinophils are the cell type most prominently interacting
with mf. Recently, strong neutrophil infiltrates adjacent to the live adult worms
were seen in untreated onchocerciasis patients, unlike those who received
doxycycline treatment, and showed drastically reduced accumulation. This

finding may relate neutrophil chemotaxis and activation to the endobacterial

12

products of Wolbachia (Brattig et al, 2001 ). T cells are distributed in a manner
suggesting that they play more of an overseer role than one of an effector cell

population that is directly anti-parasitic.

Lymphatic nodes in onchocercisiasis

O. volvulus affects lymph nodes of infected individuals. There is a range of
inflammatory responses. Acute responses are mast cell-based reactions to
cuticular collagen (Gonzalez et al 1999) In chronic stages of this disease, fibrotic
and degenerative reactions are seen. However, there is a possibility of
autoimmune responses (Eggleton et al 1999). Two general forms of presentation
exist. The first is seen in the most active, or hyperreactive onchodermatitis
“Sowda”, in which the lymph nodes are greatly enlarged with follicular
hyperplasia, activation of germinal centers, and sinus histiocytosis with varying
numbers of plasma cells, eosinophils, and neutrophils. The nodes are firm but
not painful or tender; the inguinal and femoral nodes are the most commonly
affected. Histologically, these lymph nodes are active in the cellular and humoral
arms of the immune response. The second general form is as shrunken, hard
feeling and fibrous lymph node or group of lymph nodes. These are common in
older patients with a long history of infection and chronic degenerative dermal
disease (atrophy, depigmentation, etc). Histologically, these lymph nodes are
replaced by sclerosing changes and are immunologically quiescent. Sowda
patients often present with swollen, edematous legs resembling elephantiasis,

but these changes are mostly reversible (WHO, 1987).

13

Ocular onchocerciasis

The development of ocular lesions correlates with the degree and duration
of infection (F uglsang & Anderson, 1977). In patients with palpable nodules,
serious pathology of the posterior segment of the eye was found twice as
frequently as in onchocerciasis patients without nodules (Berghout et al, 1987).
Both the anterior and the posterior chamber of the eyes are likely to be affected
by the parasite, resulting in diminution of visual acuity or complete loss of vision.
Corneal inflammation (keratitis) is a major cause of visual impairment in O.
volvulus infection. Two distinct forms of corneal diseases are recognized:
reversible punctate keratitis (snowflakes opacities) and the irreversible more
severe sclerosing keratitis, which has a permanent effect (WHO, 1987). There
are geographical differences in the prevalence of onchocercal blindness.
Blindness is more common in the Savannah areas of West Africa (2-15% of the
population of hyper-endemic areas) than in the rain forest areas of West Africa
(blindness < 2%). There is evidence for genetic variations amongst O. volvulus
(i.e. parasite variants) associated with variations in virulence, i.e. in the ability to
experimentally infect rabbits (WHO, 1987). Furthermore, when using DNA
classification techniques, it was found that there is a strong correlation between
disease severity and strain of parasite i.e. pathogenicity is strain related
(Zimmerman et al, 1992).

It has been postulated that host immunological responses could account
for ocular pathology (Bryceson, 1976). Recently, evidence has become available

that antigen-specific T cell and antibody responses are essential for the

14

development of O. volvulus keratitis and the sequence of molecular and cellular
events leading to migration of inflammatory cells to the cornea, thus leading to
the loss of corneal clarity (Pearlman & Hall, 2000). The ocular disease in this
infection may have an autoimmune component, because patients continue to
show chronic, low-level, progressive pathologic changes of the retina and retinal
pigment epithelium, even after chemotherapy to reduce parasite load. In addition,
progression of the disease of the retina and optic nerve, unlike that of the cornea,
does not appear to be related to microfilarial worm burden (Semba et al, 1990).
Molecular mimicry or immunologic cross-reactivities between host and bacterial
or viral antigens have been suggested to have a role in the development of a
number of autoimmune diseases and has been also suggested to play a role in
the development of ocular onchocerciasis. The O. volvulus antigen Ov39 is
cross-reactive with the retinal antigen hr44 and induces ocular inflammation in
rats after immunization (McKechnie et al, 2002). It was also indicated that CD4
(+) cell lines specific to the antigen Ov39 can induce ocular inflammation in naive
rats and suggested that recruitment of CDB (+) T cells may play a regulatory role,
the inflammation however, is milder than that produced by immunization. They
concluded that the absence of antibody responses to hr44 in the animals
receiving the T-cell lines might indicate a role for antibody in the development of
ocular onchocerciasis. Other human factors, such as ecological, nutritional, and
multiple infections with other parasites, may also influence the clinical

appearance of onchocerciasis in different geographical areas.

15

Immunology and immunopathology:

The pathologic manifestations of O. volvulus infections result not only from
the parasite but also from the magnitude and quality of the host immune
response (Mackenzie et al, 1985). The existence of immunity to helminth
infections in humans is a highly debatable issue. Protective immunity, acquired
and specifically directed toward filarial parasites, can be demonstrated in a
variety of experimental animal models (Grieve et al 1988; Hyashi et al, 1989).
However, it is not easy to induce protective immune responses to O. volvulus in
laboratory experimental animals and only limited success has been achieved in
this area (Naduri and Kazura, 1989). The existence of immunity to O. volvulus
infection in humans is inferred from the presence of a small number of uninfected
individuals in hyperendemic areas. Immunological characterization of such
individuals has been pursued (Nutman eta/1991) by determining antigens that
might elicit antibody responses responsible for protection. Integrating clinical and
epidemiological information with polymerase chain reaction (PCR) and ELISA
data, the immune response in putatively immune individuals was found to
correlate with a low titer of specific lgG, lgG subclasses, and lg E compared to
infected individuals (Elson etal, 1994). Nevertheless, the putatively immune
individuals were found to produce significantly more IFN gamma to O. volvulus
antigen than did those in the infected group, and less lL-10 spontaneously (Elson
et al, 1995). Thus protective immunity to onchocerciasis may be mediated in part
by an antigen-specific Th1 type response. However, some other reports relate

the immunity to the production of lL-5 and the resulting increased eosinophilia

16

(Hogarth et al, 1998). T helper type 1 (TH1) and type 2 (TH2) cells represent
terminally differentiated effector cells characterized by different cytokine
production and homing capacity. The generation of either type of response can
confer protection against pathogens or lead to immunopathology. TH1 and
TH2 polarization is a stochastic process, which is promoted by interleukin 12 (IL-
12) and lL-4 (Trinchieri 1995) Other factors that contribute to the TH1-TH2
balance are the dose of antigen, strength of antigenic stimulation, duration of T
cell receptor (TCR) engagement and the nature of co-stimulatory molecules.(Bird
et al, 1998; Gett & Hodgkin 1998; O’Garra, 1998). The balance between TH1-
TH2 cytokines regulates human immune responses to infection. lL-4 and lL-10
regulate antibody production and can suppress cell-mediated immune responses.
Distinct type 1 and type 2 T helper cytokines cross regulate the expression
and magnitude of O. volvulus-specific cellular responses in humans (Soboslay et
al, 1999). However, recently Doetze et a/ (2000) suggested that the antigen-
specific cellular hyporesponsiveness in a chronic human helminth infection is
mediated by T helper 3/ T helper 1 -type cytokines but not by a T M to Th2 shift.
Nevertheless, the TH1 pattern of cytokine production has long been associated
with immunity or resistance to helminth infection. A Th2 or type 2-cytokine
pattern has been associated with the progressive forms of O. volvulus infection in
humans (Nutman & Modlin, 1993; Johnson et al,1998; Soboslay et al, 1999;
Turaga et al, 2000). Recently, Graham et al 2001, using the bovine O. ochengi

model, suggested that neither a classical Th2 response nor a simple Th1 to Th2

l7

switch is sufficient to explain the immunomodulation associated with a patent
Onchocerca infection.

Natural killer (NK) cells are viewed as an important component of innate
resistance against a variety of pathogens. NK cells secrete IFN gamma and have
the capacity to activate macrophages before the induction of the antigen -specific
T- cell response. Therefore, NK cells might play a role in the balance of Th1
versus Th2 (Bancroft, 1993; Biron etal, 1999)

The immune attack on mf usually leads to onset of clinical symptoms
(Piessens & Mackenzie, 1982; Mackenzie et al, 1986). The clinical changes seen
in human onchocerciasis are believed to be associated with the destruction of the
microfilarial stage of O. volvulus in skin and eyes. The interaction between the
immune system and O. volvulus that on the one hand allows the survival of the
parasites for long periods of time, at other times promotes the death and removal
of parasite. The diversity in clinical presentation of onchocerciasis is considered
to reflect the intensity and quality of immune responses to the parasite or its
products. The immune responses of the host are suggested to play a crucial role
in the pathology of onchocerciasis which is usually localized to the sites where
microfilariae are attacked in the skin, eye, and subcutaneous tissues (Mackenzie
et al, 1985). The demonstration of antibodies to retinal —S-antigen (S-Ag) in
onchocerciasis patients (Chan et al, 1987; Mackechnie et al, 1993) suggests the
possibility of auto-immunity in blinding disease.

Eosinophilia and elevated serum lgE are immunological hallmarks of

infection with parasitic helminths. Immediate hypersensitivity reactions are

18

characterized by the presence of IgE antibodies, eosinophils, mast cells and
basophils, all of which have been implicated in resistance to infection as well as
pathogenesis. Eosinophils are usually attracted to infection sites leading to
macrophage activation and mf killing (David at al, 1982). The level of circulating
lgE does not necessarily give complete information about the biological function
of this immunoglobulin class and information gained from specific tests such as
measuring the release of mediators from basophils and mast cells is usually
more useful. Using histamine release measurement, it was found that sensitized
basophils might play a role in the pathogenesis of onchocerciasis. The
destruction of microfilariae was reported to involve eosinophils acting through
antibody binding (Green 1980; Mackenzie et al, 1980), and is related to papular
eruption in vivo and to punctate keratitis lesions in the eye (Mackenzie et al
1985). The antibodies involved in this killing phenomenon were thought to be
specific lgG type (Ghalib et al 1985; Ngu et al 1989). Patients with low skin
microfilarial loads have high lgG3 and low lgG4 titers, suggesting a protective
role for lgG3 and a suppressive one for lgG4 (Dafa’Alla eta/1992). Moreover,
serum concentrations of lgE and lgG are significantly elevated in sowda patients
(Brattig etal, 1987).

lL-4 and IF N gamma play crucial roles in the regulation of lgE responses
in onchocerciasis patients. lL-4 is associated with higher levels of lgE production,
while IF N gamma has been found to down regulate the lg E. The amount of lgE
produced depends on the relative quantity of lL-4 and IFN gamma secreted by

parasite- stimulated T cells (King et al, 1990). Furthermore, the induction of

19

lg E by filarial antigens depends on the concentration of the antigens. Higher
antigen concentrations suppress IgE production and this suppression can be
reversed using anti IF N gamma antibody (King et al, 1990). The mechanism
responsible for the induction of lgE by lL-4 appears to involve the regulation of
isotype switching to lgE in uncommitted B cells (Bergstedt-Lindqvist et al, 1988).
Eosinophilia has also shown to be regulated by T cells and human lL-5 is a
potent stimulus for eosinophil proliferation in vitro (Limye et al, 1991). In vivo
eosinophilia was found to be preceded by increased production of lL-5 following
microfilaricidal therapy. lL-5 secretion is sustained during the periods of
microfilarial disappearance (Limye et al, 1993). IL-5 producing cells depend upon
lL-2 for proliferation (Steel & Nutman, 1993). IL—6 was found to be elevated in
onchocerciasis patients and correlates with the occurrence and severity of post-
treatment clinical symptoms. IL-6 levels appear to depend on the intensity of
infection (Ali, unpublished). Serum TNF levels are also elevated after treatment,
but did not correlate with the infection intensity or reaction severity (Turner,
1994)

Defective responsiveness of peripheral blood lymphocytes in vitro from
onchocerciasis patients has been reported (Green et al, 1983, 1985; Gallin et al,
1988; El Khalifa et al, 1991; Soboslay et al, 1992). The major conclusion from
these studies is that responses to parasite antigen in patients with the
generalized form of onchocerciasis are minimal, compared to putatively immune
persons living in (an )endemic areas without active infection. They were also

found to have higher lL-2 and IFN gamma responses to onchocercal antigens

20

compared to patients with the generalized form of onchocerciasis (Ward et al,
1988; Soboslay et al, 1999). However, there is less published information on
cytokines in onchocerciasis patients representing the varied clinical spectrum.

In a murine model, CD4 (+) T cells mediate sustained O. volvulus keratitis by
regulating eosinophil recruitment to the cornea (Hall et al, 2000). This may be
due to the mobilization of blood and tissue eosinophils during clearance of
infection. Previous work indicated that, CD4 (+) Th2 cell namely, lL-5 was found
to be associated with clearance of infection (Hogarth & Bianco, 1999). However,
it could also lead to pathology. Flow cytometric determination of lymphocyte
subset distributions show improvement in the CD4 (+) T-cells status of patients
after treatment to reduce microfilariae with ivermectin. Production of lL-2 and lL-4
by lymphocytes induced by phytohemagglutinin (PHA) increased one month after
treatment (Freedman et al, 1991). Treatment with ivermectin seems to enhance
cellular proliferative response 36 months post-treatment (Steel& Nutman, 1993).
However, a generalized suppression in lymphocyte proliferation has been
observed 2 weeks after treatment (Nutman, 1987), probably resulting from the
release of suppressive microfilarial antigens (Lal et al, 1990). Several findings
support the hypothesis that microfilaria-derived products are important in
suppressing lymphocyte proliferative responses to O.volvulus antigens.
Furthermore, ivermectin has immunostimulatory properties that are associated
with altered functions of T-lymphocytes, particularly T-helper cells (Blackley et al,
1991). Parasite antigen-specific deficits in lL-2 and lL-4 production were not

reversed by ivermectin treatment, leading to the suggestion that some

21

components of non-reactivity are long lasting and are not affected by diminished
microfilariae in detectable numbers in skin tissues. Further work is needed to
better understand the effect of ivermectin treatment on the host immune
response. The interplay between infection status and the balance between

immune responsiveness and immune modulation also remain to be addressed.

Diagnosis

Filariasis in general continues to present diagnostic challenges. The broad
spectrum of clinical manifestations associated with this infection creates a
diagnostic paradox. The accurate and specific diagnosis of filarial infection has
become increasingly important as a mean of monitoring the efficacy of mass
distribution of Mectizan. This goal has been hampered by our inability to

distinguish between past and current infection.

1. Parasitological diagnosis of infection

The detection of unsheathed microfilariae in the skin involves removing
small snips of skin (Consistent i.e. almost of the same size bloodless biopsy) and
incubating them in an aqueous solution, preferably (RPMI 1640), to allow
microfilariae to migrate out so that they can be observed microscopically. A
bloodless skin snip weighing from one to several micrograms can be removed
quickly and without much discomfort, is obtained using a razor blade, or Walser-
type corneoscleral punch. This is more convenient and allows uniformity in snip

size. Microfilariae will emerge from the sample within 4 hrs, and the intensity of

22

infection is reflected in the number of microfilariae emerged from the snip (Taylor
et al, 1989). Sowda or severe reactive onchodermatitis patients have few or low
microfilarial load of viable microfilariae, or may even show a negative test by this
method. Skin from the pelvic girdle provides the best chances for detection
(Williams et al, 1985 a, b).

2. Ultrasonography:

Onchocerciasis can also be diagnosed by detecting microfilariae in the
eye by using the slit- lamp. Detection of subcutaneous nodules is also used for
clinical diagnosis and the introduction of ultrasound facilitated the detection of
these nodules (Homeida et al, 1986; Leichsenring et al, 1990). Onchocercal
nodules must always however, be distinguished from other similarly presenting
lesions such as lipomas, foreign bodies, granulomas, and sebaceous and
dermoid cysts.

3. Immunological and Molecular Diagnosis:

The Mazzotti test, which is an allergy- based reaction, was frequently used
as a diagnostic aid for patients with negative skin snips. It is (carried out done by
administration of 0.5 mg DEC orally every 4-6 hours for three days and then 1
mg DEC every 4-5 hours. Dermal changes were seen, consisting mainly of
papular responses (El Shiekh, 1985). New procedures have been developed for
the immunodiagnosis of onchocerciasis and these will be of great significance
since the detection of microfilariae by using skin snip is not feasible in prepatent
infection or in those with strong immunological responses. A wide variety of

antigens have been utilized in this procedure, including soluble O.volvulus crude

23

antigen. Also, recombinant polypeptides, a cocktail of three recombinants have
been field tested for use in O. volvulus infection (Ramachandran, 1993). It was
found to detect 0. volvulus infection during the prepatent period. These antibody-
based assays, however, cannot reliably distinguish past and current infection, nor
have they been helpful in following individuals after chemotherapy. A PCR test
for skin snip samples to detect the presence of O. volvulus DNA in order to
rapidly assess the epidemiology of the disease in endemic areas (Zimmerman et
al, 1994) was found to be more sensitive than standard skin snip examination.
Circulating antibodies are detectable using an ELISA system with blood obtained
from finger prick and collected onto filter paper. This test is useful in those under
15 years and could be integrated with other disease programs that monitor blood
samples, like malaria (Botto et al,1999).
4. Epidemiological Diagnosis

Diagnostic procedures in onchocerciasis are used to determine the
prevalence of infection, to identify individuals requiring treatment, to evaluate the
success of treatment, and to assess the impact of control efforts. A non-invasive
epidemiological technique is needed for the detection of disease to determine
eligibility of mass treatment programs, as has been launched by the WHO in the
African Program for Onchocerciasis Control (APOC). This technique is known as
Rapid Epidemiological mapping for Onchocerciasis. It comprises a rapid search
for nodules in individuals suspected to have the infection. It requires experience
and knowledge for the personnel who perform the technique. It greatly helped to

estimate endemicity in order to start the control program.

24

Treatment

Treatment of onchocerciasis has been a problem in the past and is not yet
satisfactory. Ideal treatment of infection with O. volvulus would include drugs that
kill the adult worm (macrofilaricidal) and the microfilariae (microfilaricidal), with
minimal side effects on those who receive this treatment. The history of treatment
involves suramin, diethylcarbamazine (DEC) and recently ivermectin (Mectizan),
which is currently the drug of choice. Suramin was administered intravenously in
repeated doses, as a macro— and micro-filaricidal, but the drug had severe
cumulative toxicity because of its very slow excretion. DEC was administered
orally in a multiple dose regimen(s), but was associated with a very high increase
of adverse reactions, resulting from the rapid killing of microfilariae in the skin,
subcutaneous tissues and the eyes. These can lead to life threatening severe
allergic-like clinical responses, including severe itching, lacrimation, urticarial
oedma of the skin, swelling and tenderness of the lymph nodes, maculopapular
eruption, pyrexia and sometimes severe postural hypotension (El khalifa et al,
1985; Awadzi et al, 1980). In the patients with ocular lesions, DEC aggravates
the condition and can cause complete loss of vision (Bird et. al, 1980). Thus I
patients treated with Suramin or DEC should be hospitalized or treated under
close medical supervision.

In view of these problems there was a desperate need for an alternative
therapy for onchocerciasis. lvermectin is a semi-synthetic macrocyclic lactone

produced by the actinomycete Streptomyces avennititis sp. developed by (Merck

25

and CO., Inc.) it has been extensively used in veterinary medicine for treating
internal and external parasites (Campbell, 1985). The drug was introduced for
human onchocerciasis (WHO, 1987). It was a revolutionary breakthrough, as it
has an effective microfilaricidal action and it clears microfilariae from the skin with
minimum side effects (Aziz et al, 1982; Awadzi et al, 1980; Campbell 1991). The
drug is given in a single dose and produces a prolonged reduction in microfilarial
loads. It has provided for the first time a feasible chemotherapy for large-scale
treatments. It has been used successfully in mass treatment programs (Baraka et
al, 1995). It improves skin lesions except for depigmentation (Pacque et al,
1991), improves visual acuity, and reduces the source of infection (Newland ef al,
1988). The drug was also found to be safe even in individuals with the severe
ocular form of the disease. Recently, a model for human onchocerciasis (O.
ochengi infection in cattle) demonstrated the effectiveness of administering the
antibiotic tetracycline together with ivermectin, resulting in a macrofilaricidal
effect. This response is hypothesized to be related to the action of tetracycline on
Wolbachia endobacteria; abundant in O. ochengi (Trees et al, 2000) this could be
a useful procedure that may increase options for the control of human
onchocerciasis. Recent findings support a role for Wolbachia products in
mediating the inflammatory responses seen following treatment of
onchocerciasis and suggest new targets for modulating these reactions (Keiser
et al, 2002)

lvermectin uptake was thought to be mediated by specific high affinity

receptor. The interaction between ivermectin and the receptor leads to the

26

release of gamma aminobutyric acid (GABA) from the nerve ending and enhance
the binding of GABA to its receptor leading to direct increase in the membrane
permeability to chloride ions (Campbell, 1985). lvermectin is suggested to have a
significant effect in the release of microfilariae from the female gravid uterus by
reducing the number of the multicellular embryogenic stages in worms exposed
to multiple does of ivermectin. This may be partially due to the reduction in the
effectiveness of insemination in female worms and minor impairment of
oogenesis (Chasse et al, 1993). Moreover, ivermectin may have
chemoprophylactic effects, which might contribute to the maintenance of low
microfilarial production in areas of on-going transmission (Klager et al, 1993).
However, recently some recurrence was observed in a small proportion of
treated patients with ivermectin after only one month of treatment, unlike
previous reports (Ali, see chapter three, Acta Tropica, in press). This suggests
that at least for the treatment of a small subset of individuals, Mectizan may need
to be administered more frequently than once a year, perhaps as often as every
three months. This also raises the possible involvement of the immune
responses in microfilaricidal mechanism of action of ivermectin. Studies
concerning immunological responses after repeated doses of ivermectin in
patients with onchocerciasis emphasized the apparent long term safety of
ivermectin through the demonstration of the absence of immunopathological
responses induced by repeated ivermectin treatment (Steel et al, 1991). The
immunosuppression due to the disease is reversible after ivermectin treatment

(See chapter one; Ali submitted); there is a sustained production of lL-2 and IF N

27

gamma, which contribute to O. volvulus infection. Thus ivermectin may be
needed to enhance immunity against onchocerciasis (Soboslay, 1994). It might
also be involved in the mechanism of killing by the drug.

Recently, the African Program for Onchocerciasis Control (APOC),
operated by WHO, adopted a control strategy for combating onchocerciasis,
which relies mainly upon ivermectin treatment. This new approach employs a
community directed treatment strategy to secure the issue of sustainability. As
the drug will only kill the microfilarial stage, treatment should be repeated at least
once a year for at least 12 years, the expected life span of the adult worm. The
program started in nineteen African countries and has treated more ten million
individuals.

The present contraindication criteria for ivermectin treatment include
pregnancy; age <5 years or wt <15 kg and breast-feeding women up to one week
after birth. Sickness in individuals or concurrent illness should also be borne in
mind in the treatment campaign. The involvement of the central nervous system
(CNS) in the pathogenesis of the infection needs to be considered especially the
life threatening adverse reactions to ivermectin treatment of patients infected with
the filarial parasite Loa Ioa (Ducorps et al 1995). Currently, with the introduction
of the Mectizan-Albendazole program for lymphatic filariasis, the mechanisms for
handling and minimizing the problems where the two diseases are co-endemic is

considered essential part of control program management.

28

In conclusion, ivermectin alone has been extremely successful so far in
the aim of eliminating onchocerciasis as a public health problem. However,
elimination of transmission has proved to be more difficult.

It is worth noting that research is going on to identify a new drug that can
kill adult worms without harmful side effects. Current studies are focused on the
potential of moxidectin as a macrofilaricidal, the potential for existing antibiotics
as alternative treatments for the elimination of Wolbachia as a target for drug
development; and the development of methods for detection of ivermectin
resistance.

Nodulectomy

Surgical removal of nodules from accessible parts of onchocerciasis
patients helps in reducing the parasite burden in the body and hence decreases
the severity of the disease. But many nodules are not easily detectable. This is
not a feasible way of control strategy as it is very cost ineffective, and needs
hospital support which is not available in most situations or cases of the remote

areas where the disease prevails.

29

Rationale and Objective

Onchocerca species induce both cellular and humoral immune reactions.
The intensity of these reactions varies considerably with the clinical
manifestations of the infected hosts. Infections are chronic, and persistence of
the parasites for several years argues for highly adapted mechanisms of immune
evasion. Cellular responses to parasite antigens are suppressed in individuals
with high parasitemia “generalized onchocerciasis”, unlike in patients either free
from infection, or those manifesting localized disease with very low live
microfilarial loads if any. The mechanisms that control the levels of circulating
microfilariae in vivo are not well understood. Speculations to the process of in
vivo killing of the microfilariae have been made. Onchocerciasis persists in most
of the treated patients who continue to live in endemic areas. A degree of
resistance to the infection may exist. Many questions in onchocerciasis
concerning the interplay between infection status and the balance between the
immune responsiveness and immune modulation remain to be addressed.

Microfilaricidal drugs are thought to somehow involve the immune system
in their killing mechanism. It has been proposed that their action involves
unmasking previously hidden parasite antigens and thus stimulating antigen
presentation to the effector cells.
This study is an attempt to gain better understanding to the role of the immune
system in the development of pathology and varied clinical presentations seen in
onchocerciasis patients. It is also intended to identify any role the immune

system might play in the microfilaricidal action of ivermectin (Mectizan).

30

Objectives:

1/To characterize humoral and cellular immune responses in onchocerciasis
patients with different clinical presentations

2/ To study the effect of ivermectin treatment in these patients and to see if the

immune system mediates the outcome of treatment.

31

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47

Chapter Two

Immune Responses Against Microfilariae are Associated with the Severity

of Onchodennatitis and Treatment History

This Chapter has been presented at the American Society of Tropical Medicine 8

Hygiene (ASTMH) 50th meeting

48

INTRODUCTION

Onchocerciasis is a filarial infection characterized by a wide variety of
ocular and dermal pathology, the latter ranging from minor irritation to severe
reactive dermatitis and in many cases includes visual loss. The basis for clinical
variations in this disease is believed to primarily involve an intimate relationship
between the parasite and the host’s immune system (Mackenzie et al,
1980,1985, 1987). The interaction between the immune response, the major
pathology-inducing event (microfilarial destruction), and other specific events that
lead to this parasite-induced damage in all likelihood lies at the basis of all of the

clinical presentations of this varied and complex disease.

Onchocerca volvulus microfilariae (Mf) have long been known to be
involved in the acute papular dermal responses and the typical punctate keratitis
seen in the corneas of affected people (Mackenzie et al,1985, Williams et al,
1987). It has also been assumed that the worsening clinical presentation of
chronic papular dermatitis and other more severe changes seen in
onchodermatitis involve inflammation related to microfilariae and their
destruction. Although other theories have been suggested as contributory to the
pathogenesis, autoimmunity and secondary infection to name two, the most likely
hypothesis is that increasing cycles of inflammation involving microfilariae are

central to all these clinical reactions (Mackenzie et al, 1985).

49

Specific antibodies are generated against many O.volvulus antigens and
various studies have been directed at describing them (Mackenzie et al, 1985;
Green et al, 1981; Garate et al, 1996; Gallin et al, 1995; Wani, 1997). Antibodies
specifically involved in the destruction of O.volvulus microfilariae through
antibody-dependent cell cytotoxicity mechanisms have been described
previously (Green,1981; Mackenzie et al, 1980; Williams et al, 1987). Cell
mediated immunity (CMI) has also been described as being involved, and studies
on certain clinical forms of the disease such as reactive onchocercal dermatitis
(“sowdh”) are known to involve CMl (Bartlett et al, 1978; Green et al, 1985;
Elkhalifa et al, 1991; Mackenzie et al, 1985; Baraka et al, 1995; Darge et al,
1995). It is assumed that Lin/iii) cellular responses to adult worm antigen
cocktails also includes activities stimulated by microfilarial stage antigens, as

microfilariae are a significant component of adult female worms.

In the current study we investigated the significance of these immune
responses directed against microfilariae in onchocerciasis patients presenting
with two major forms of dermal disease: mild or asymptomatic, and severe
dermal manifestations. We examine these immune responses to determine the
significance of reactions to O.volvulus microfilariae in the pathogenesis of

disease in these two clinical groups.

50

Patients and Methods
Study populations:

The patients examined in this study became infected in either of two
Sudanese onchocerciasis endemic areas; the south west (Bahr el Ghazal) or the
Equatorial regions of Southern Sudan (Bryant et al., 1935; Mackenzie et al.,
1987). They were displaced to the North due to civil unrest and settled in camps

around the capital Khartoum.

The clinical classification of patients (n=28) in terms of onchocercal
dermatitis was carried out using the schemes developed by Mackenzie et al.
(1985) and Murdoch et al. (1993). The patients were grouped in two categories:
Group A - mild (minor acute change) or asymptomatic (no obvious dermal
change) onchodermatitis (n=12), and Group B — Severe (active and chronic skin

changes) onchodermatitis (n=16).

A subgroup of nine patients (seven from group A and two from Group B)
was studied after they had been treated with therapeutic doses of Mectizan®
(150ug/Kg). Control sera and cells, obtained from a group of individuals who
were resident outside the areas endemic for this disease and who had never
been diagnosed as being infected with O. volvulus. were used in cytoadherence

assays (n=2) and stimulation index (n=5).

51

Parasite antigen preparation:

Nodules from Sudanese patients were digested with collagenase (Schultz-
Key et al. 1977), and the released adult worms homogenized using a tissue
grinder; insoluble material was removed by ultracentrifugation. Soluble
O.volvulus antigen (OvAg) was passed through a 0.2micrometer syringe filter

and the protein concentration was adjusted to 1mglml (Lowry et al, 1951 ).

Microfilariae:

Two onchocercal nodules were surgically removed from patients and kept
in RPMI-1640 media on ice for transport to the laboratory, cut into small pieces
using sterile surgical blades and the Mf were allowed to emerge. The medium
containing released Mf was collected and transferred to sterile petri dishes (Ngu
et al, 1981 ). Equal volumes of 0.80% agarose dissolved in RPMI-1640 medium
by heating and cooled to 40°C were poured into the petri dishes and the plates
then carefully rotated to mix the microfilariae suspension with the agarose. The
solution was then allowed to solidify at room temperature. 10 ml of sterile RPMI-
1640 medium were carefully layered on the agarose surface and the plates were
incubated at 37°C for 40 min. Live Mf that had migrated to the RPMl-1640
overlay were aspirated into 15 ml centrifuge tubes and centrifuged at 1000 rpm
for 3 min, the media decanted and the Mf washed twice with RPMl-1640 by
centrifugation as above. Mf were then finally suspended in RPMI-1640 medium
containing 10% (vzv) heat inactivated fetal calf serum, counted and adjusted to

1000 mf/ml.

52

Cells:
Eosinophil Rich Population for Cytoadherence

Heparinized blood (20 ml) was collected from onchocerciasis patients
whose white blood cell differential count was >43% eosinophils. 1/6 (vzv) of 4.2%
dextran in phosphate buffered saline (PBS) was added to the blood in a
centrifuge tube and mixed well and incubated at 37°C for 40 min in an upright
position. The superficial cellular layer was then carefully aspirated into RPMI
1640 and the cells pelleted by centrifugation at 1200 rpm for 10 min, washed

twice in RPMI-1640 and finally resuspended for use at 4x106 cells Iml.

PBM Cells for Transformation Cultures

Whole blood was obtained from each subject, and peripheral blood
mononuclear cells (PBMC) were separated by centrifugation over a gradient of
F icolI-Hypaque (Histopaque 1077, Sigma Corp.) and adjusted after washing to
the required cell concentration. PBMC were suspended in RPMI-1640 (Sigma)
supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 2
m ML-glutamine, 1m M HEPES, 0.04 mM 2-mercaptomethanol, and 80

microgram of gentamycin, washed and counted.

Assays
Cyto-adherence Test:
Fifty microlitres of Mf suspension (containing 50 Mf) were pipetted into the

wells of 96 well flat bottom tissue culture plates. The same volume of undiluted

53

serum from each patient or control was added to duplicate wells. The plate was
then incubated at 37°C for 1 hour to allow the antibody to bind to Mt. 100
microlitres of eosinophil suspension (4x10'5 cells) were added to each well.
Adherence was defined according to an estimation of the area of the parasite’s

surface covered with cells. The following scale was used as a guideline:

0 = none, or only a few cells adherent

1 = up to 25% surface covered with adherent cells
2 = 25-49% surface covered

3 = 50 — 74 % surface covered

4 = 75% to completely covered

Cultures were monitored, using an inverted microscope, after 3 hr incubation;
and they were also checked at 24 hr to ensure no false negative assessments
had occurred. At least 30 Mf were assessed before recording the adherence

score for a particular culture.

In - Vito blastogenesis

Blastogenic responses of (PBMC) to mitogens and antigens were
measured using a [H3] thymidine incorporation technique. 1x10 5cells in 100ul
RPMI 1640 were pipetted into the U-shaped wells of a 96 microtitre tissue culture
plate (Corning, NY). Cells were stimulated by 10 ul of phytohemagglutinin (PHA)
mitogen (Sigma ) at 5 ug/ml or with Ov Ag at 20 uglml. The cells (3x105 lwell)
were distributed in triplicate into 96-well round-bottomed microtiter plates (Nunc

AIS, Roskilde, Denmark). The cultures were incubated for 5 days at 37°C in a

54

humidified atmosphere of 5% C02 in air, with a final volume of 200 ul/well, in the
presence of 20 ug/mI phytohemagluttinin (PHA - Pharmacia, Upsala,Sweden), or

for specific stimulation, with 20 ug/ml of OvAg.

Eighteen hr before harvesting, 1 Ci of [3H]-thymidine (Amersham
International, Amersham,UK) with specific activity of 5 Ci/mmol, was added to all
wells, including negative controls i.e. those without antigen or mitogen. Cells
were harvested on fiber filters (Titertek, Flow Laboratories Inc.) Incorporation of
[3H] thymidine was measured in a beta-scintillation counter (Becton Dickenson
Ltd). Results were expressed as the stimulation index (SI) derived from the
counts per minute (cpm) of the average of three replicate wells containing
antigen minus the background counts of non-stimulated wells; results are

expressed as counts x 10'3.
Statistics

Groups were compared using the SSPS program and significance

determined using Student’s t test, the paired “t” test and Pearson Correlation test

55

RESULTS

Clinical Presentation and Parasitology

Twelve patients (Group A) had a dermal presentation of essentially normal
skin or with minor acute papular reactions. Sixteen individuals (Group B) had
severe onchodermatitis typified by chronic indurated papules, extensive pigment
variation, thickening of the epidermis, edema of the dermis and severe persistent
pruritus. O.volvrgis microfilarial loads were highest in the mild skin disease group
(mean of those measured = 27.7 +I12.9), whereas markedly lower microfilarial
loads were evident in the severe skin disease group (mean of those measured =
7.9 +l- 5.5). Thus there was an inverse relationship between the microfilarial
count and the severity of clinical manifestation (Table 1). The microfilarial loads
in Group A were significantly more than those of Group B (p>000.1) as

determined by Student’s t test.

Antibody Mediated Responses

The two groups differed in the ability of the serum to mediate eosinophil
adherence to the surface of microfilariae also showed a difference between the
two groups. Patients from the mild dermatitis group showed minimal positivity
(average = +) in the cell adherence assay (Table 1), whereas sera from those
with severe dermatitis mounted strong responses in the cell adherence assay
(average = +++). The adherence system was optimal at 3 hr, and observation at

24 hours did not reveal any late developing adherence reaction.

56

Cell Immune Responses

Cell proliferation responses to O.volvulus parasite antigens (0v Ag) -
(Table 2) were higher (12.3 +I- 1.9) in patients with more severe skin
manifestations (and lower Mf loads). The proliferative index in the mild
dermatitis group was 2.9 +/- 0.6. This difference was statistically significant (p=
>001). The responses to OvAg in control individuals (4.5 +/- 0.4) were similar to
those in Group A (those with mild skin disease). PHA responses were measured
with one group (Group A): these are within the range expected and indicate that
the culture system being used was technically adequate.
Associations between Antibody and Cell Mediated Immune Responses:

A positive relationship between the presence of the ability to mount an
active cell adherence response and an active cell mediated response was also

seen. Using a Pearson Correlation this was seen to be a significant relationship.

Post Mectizan® Therapy

The cell-mediated responses of 9 patients (7 from Group A and 2 from
Group B) were assayed after treatment with therapeutically with ivermectin and
were increased in all cases, significantly in those with mild disease (Group A).
This was more obvious in the mild/asymptomatic patients (average increase >
2.5 X) than in the two severe dermatitis patients, in which the increase was

minimal (Figure 3).

57

Discussion

It is well accepted that the pathological changes in onchocerciasis are
related to the microfilarial stage of this nematode; however, it has not been clear
as to the exact role of the immune system in this disease. Immune responses to
O.volvulus are well long documented (Green et al, 1981; Mackenzie et al, 1980;
1985a; 1985b) but correlation of host responses with individual patient’s clinical
status has remained uncertain at best. The results of the present study show
that two types of immune response mounted against O.volvulus antigens
(including microfilariae) differ between patients with the two major forms of
dermal presentation of onchocerciasis. This finding is in keeping with various
published studies, but is the first study that clearly identifies different immune
responses against the microfilarial stage as being directly related to the dermal

clinical presentation.

A distinct positive correlation between immune response and clinical
severity is seen. Corresponding to the increased immune response, and the
expected increase in destruction of microfilariae, is a decrease in microfilarial
load with severity of skin disease. Relatively low dermal Mf levels are seen in
individuals with clinically obvious skin disease and active pathology (group B in
this study). Very low levels of living microfilariae have been described in patients
with the severe onchocercal skin disease known as “sowda” (or reactive
onchodermatitis) (Bartlett et al, 1978; Baraka et al, 1995). This situation contrasts

with the popular belief that microfilarial loads simply increase with duration of

58

infection, and that disease severity parallels these increased parasitic loads. The
host’s ability to destroy the microfilariae is at least as an important factor in the

clinical outcome as is the duration of infection.

Individuals suffering from mild dermatopathology (group A) were less
effective than severely affected patients in promoting eosinophil adherence to O.
volvulus mf, and therefore probably less effective at destroying mf through an
antibody mediated process. This finding that may explain in part their clinically
quiescent state when compared to the severely affected group, whose serum
mounted stronger responses against the worm surface. Adherence correlates
with clinical manifestations associated with microfilarial destruction (Mackenzie et
al, 1985). Likewise, observations on patients who were recently treated with
ivermectin showed immunological signs of increased responses against these

parasites.

The sequence of events and consequences associated with the death,
breakup and removal of microfilariae are still open to conjecture. Immune, non-
specific and drug-induced pathways can all occur at different times in this
disease. This present study emphasizes the immune component and suggests
that both antibody and cell mediated immunity are involved. Other studies have
shown that the antibody response to microfilariae varies with time in an episodic
manner (Mackenzie et al, unpublished). The death of microfilariae commonly

induces a local inflammatory reaction (Mackenzie et al, 1985) and when killing is

59

induced by chemotherapeutic agents such as diethylcarbamazine severe
anaphylactoid Mazzotti reactions can occur; these occur less with the now
standard microfilariacidal agent for onchocerciasis, Mectizan®. Control of the
associated pathogenic side reactions is important, especially in those individuals
who react severely. Understanding the role of the immune response in the
induction of these reactions, and the extend to which this response accounts for

the drug effect is important.

lvermectin appeared to enhance the cell-mediated responses in
onchocerciasis patients when examined one to three months after treatment.
This suggests that the immune system plays a significant role in chemotherapy if
not in the initial induction of treatment, then at least in maintaining the ongoing
mf destruction. The consequences of altered immune responses for the
effectiveness of microfilariacidal regimes deserve examination, particularly in
light of the suggestion that Mectizan® is sometimes minimally effective in certain
individuals (Ali et al, in press). There are suggestions that ivermectin is less
effective in small subset of infected individuals. This may involve a deficit in their
immune response. Our results provide support for the involvement of the

immune system in the acute and subsequent actions of ivermectin.

The finding of an inverse relationship between microfilarial load and

severity of skin disease has important implications for control programs that are

based on the assumption that microfilarial load is directly related to disease

60

status. Such programs should necessarily include those individuals with lower
levels of infection if one wants to alleviate the pain and discomfort of those
severely affected. Currently, mass drug administration programs often exclude
hypo-endemic areas (APOC, WHO) and thus may miss areas where people
suffer from severe onchodermatitis. This policy should be revisited to take
account of those most severely affected with this disease, a policy that is
incorporated into other filarial disease program strategies, such as that of
morbidity control in the lymphatic filariasis elimination program. These findings
also underline the importance of including clinical evaluations in epidemiological
assessments of disease prevalence instead of relying on parasitological

parameters alone.

61

REFERENCES

Baraka OZ, Khier MM, Ahmed KM, Ali MM et al. Community based
distribution of ivermectin in eastern Sudan: acceptability and early post-
treatment reactions.Trans R Soc Trop Med Hyg. 1995; 89: 316-8

Baraka OZ, Mahmoud BM, Ali MM, Ali MH, el Sheikh EA, Homeida MM,
Mackenzie CD, Williams JF lvermectin treatment in severe a symmetric
reactive onchodermatitis (sowda) in Sudan. Trans. R. Soc. Trop Med Hyg
1995; 89 : 312-5.

Bartlett A, Turk J, Ngu JL et al. Variations in delayed hypersensitivity in
dermal onchocerciasis. Trans R Soc Trop Med Hyg. 1978; 72: 372-377.

Bryant J. Endemic retino-choroiditis in the Anglo-Egyptian Sudan, and its
possible relation to Onchocerca volvulus. Trans R SocTrop Med Hyg
1935; 28: 523-532

Drage K, Buttner DW. lvermectin treatment of hyperreacative onchodermatitis
(sowda) in LiberiaTrop Med Parasitol 1995: 46:206-12.

El khalifa MY, Ghalib HW, Dafa’alla T 8 Williams JF Suppression of human
lymphocyte responses to specific and non-specific stimuli in human
onchocerciasis. Clin exp Immunol 1991 86, 433-439.

Gallin MY, Jacobi AB, Buttner DW, et al. Human autoantibody to defensin:
disease association with hyperreactive onchocerciasis (sowda).J Exp Med

1995;182:41-47.

62

Garate T, Conraths FJ, Hamett W, et al Identification of Onchocerca volvulus
collagen as an antigen mainly recognized by antibodies in chronic hyper-
reactive onchodermatitis (sowda).Am J Trop Med Hyg1996; 54:490-497.

Ghalib H., Mackenzie C.D. Williams J.F. Severe onchocercal dermatitis in the
Ethiopian border region of Sudan. Ann Trop Med Parasitol. 1987;81:405-
419

Greene BM, Taylor HR, Aikawa M 1981 Cellular killing of microfilariae of
Onchocerca volvuluszeosinophil and neutrophil-mediated immune serum
dependant destruction. J Immunol 127: 1611-1615.

Greene B, Fanning, M 8 Ellner,J Non-specific suppression of of antigen-induced
lymphocyte blastogenesis in onchocerca vovulus infection in man.
Clin.exp.immunol.52,259.

Lowry 0, Rosenbrough J, Farr A, 8 Randall J (1951) Protein measurement with
the folin phenol reagent. J. biol Chem. 193, 265

Mackenzie CD 1980 Eosinophil leukocytes in filarial infection. Trans R Soc Trop
Med Hyg 74 (suppl):51-57.

Mackenzie CD, Williams JF, Sisley BM, Steward MW, O’Day J 1985. Variations
in host immune response in relation to immunopathology in human
onchocerciasis. Rev Infect Dis 7(6):802-808.

Mackenzie CD, Guderian RH, Proano SR, et al. reactiva oncocercotica
(Sowdah) en la Provincia de Esmeraldas, Ecuador. Revista de Facultad
de Ciencias Medicas (Quito, Ecuador) 1985; 10: 121—125.

Mackenzie CD, Williams JF, Sisley BM et al. Variations in Host Responses and

63

the pathogenesis of human onchocerciasis. Rev Infect Dis 1985; 7: 802—
808.

Mackenzie CD, Williams JF, O'Day J, et al. Onchocerciasis in Southern Sudan:
parasitological and clinical characteristics in the Bahr El Ghazal Province.
Am J Trop Med Hyg 1987; 36: 379—382.

Mackenzie 00, Williams J.F., Guderian R.H. et al In: “Filariasis” Ciba
Foundation Series 1987; 127: 46-72. Ed. Evered D. and Clark 8. ,John
Wiley 8 Sons Chichester UK.

Ngu J, Neba G, Leke R, Titanji V, Asonganyi T 8 Ndumbe P (1981) Selective
recovery of living microfilariae from onchocerca volvulus nodules. Acta
Tropica. 38,261.

Wanni NO, Strote G, Rubaale T, Brattig NW. Demonstration of
immunoglobulin G antibodies against onchocerca volvulus excretory-
secretory antigens in different forms and stages of onchocercaisis. Trans
R Soc Trop Med Hyg, 1997: 91:226-30

Williams JF, Ghalib, HW, Mackenzie CD, Elkhalifa MY, Ayuya JM and Kron MA.
1987 Cell adherence to microfilariae of onchocerca volvulus: a
comparative study. Filariasis. Wiley, Chichester (Ciba Foundation

Symposium 127) 146-163.

64

Table 1. In vitro cyto-adherence activity of serum from onchocerciasis patients
with different clinical presentations and controls using eosinophils and
Onchocerca volvw microfilariae.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Patient status Mf/mg 3 hour incubation
Asymp/Mild (n=9)
48.7 ++
37.9 +
32.8 +
25.7 -
18.6 +
Nd ++
19.1 -
Nd -
11.2 ++
AVERAGE 27.7 +/-3.9 +
Severe (n=7)
0,9 +++
3,7 ++++
12.8 +++
8.7 ++
Nd +++
Nd +
13.4 ++
AVERAGE 7.9 +/- 2.8 +++
Control (n=2)
0 -
o -
AVERAGE -

 

 

 

 

65

Table 2

 

 

 

 

Mitogen or Mild skin group Severe skin group Negative controls
Antigen (SI) (n=12) (SI) (n=16) (SI) (n=5)

PHA 121.15 +/- 7.5 21.5 +/- 3.7 132.6+I- 8.7

0V 3.0 +/-0.6 12.3 +l-1.9 4.5 +I- 3.6
anfigen

PHA+OV 44.7+I- 9.7 ND ND

anfigen

 

 

 

 

 

Negative: never been diagnosed as onchocerciasis positive or suffered from any
corresponding disease.

66

 

FIGURE 2.
CHANGE IN IMMUNE RESPONSE AFTER TREATMENT WITH MECTIZAN®

 

 

Stim ulation Index

 

P1 P2 P3 P4 P5 P6 P7 P8 P9

Individual Patients before and after treatment

 

 

 

 

0 Individual Patients:
P1-7 = patients with asymptomatic/mild dermatitis
P 8-9 =’patients with severe dermatopathology

All tests were carried out 2 months after stande treatment with Mectizan®

67

Chapter Three

lmmunocompetence may be important in the effectiveness of

Mectizan® (ivermectin) in the treatment of human

onchocerciasis

This chapter has been accepted for publication at ACTA TROPICA

68

The microfilariacidal agent MectizanR (ivermectin) has been donated
since 1987 by Merck 8 Co. Inc., and has provided for the first time a
feasible chemotherapy for treatment and control of onchocerciasis. This
drug has been central to the ongoing efforts to combat this disease through
self-sustainable community-based treatment. Original studies showed that
annual doses of MectizanR reduced the microfilarial loads to, and
maintained them at, very low levels for 9 months to a year after a single
treatment (Awadzi et al, 1989; Brown 8 Neu, 1990; Ette et al, 1990); thus
for mass drug administration a single annual dosing regime was
recommended. As this drug does not kill adult worms it is believed that
these annual dosing programs should continue for at least 12 years, the
average of the adult parasite life span. The possibility of parasitological
unresponsiveness to Mectizan treatment or selection for drug resistance in
Onchocerca volvulus has been proposed (Grant, 2000; Boussinesq and
Gardon, 1999), and here we report from Sudan our own findings
concerning the possibility of reduced effectiveness of the drug in some
onchocerciasis patients.

In our continuing effort to better understand the effects of MectizanR
Treatment we have studied displaced people originally coming to Khartoum
from Southern Sudan. Patients with onchocerciasis are usually expected to
receive parasitological and clinical relief from Mectizan® (150

microgram/Kg-body weight) for at least 10 months after their initial

69

treatment; suppression of pruritus usually occurs for at least 12 months
(Brieger et al, 1998). A group of 47 patients reported back to our clinic
unexpectedly well before this time period complaining of a recurrence of the
same intense pruritus that had affected them before the initial treatment.
This pruritus was clearly distinct from the post treatment reactions often
seen shortly after administration with this drug. A second group of 12
treated patients responding in the manner that is normally expected after
treatment, i.e. suppression of pruritus for at least and usually longer than 10

months, were also studied for comparison.

Entry of individuals into the Mectizan ® treatment program required
a baseline clinico-parasitological workup, including microfilariae (mf) loads in skin
of the iliac crest region as assessed by the standard skin snip technique and the
recording of any onchocercal nodules detected by palpation. Cellular immune
responses to Onchocerca volvul_us_ , namely antigen specific cell transformation
of blood lymphocytes from these patients were carried out in standard
lymphocyte - thymidine incorporation assays using parasite antigen derived from
0. volvulus. Cells isolated on Ficol Hypaque gradients were cultured in RPMI-
1640 supplemented with 10% heat-inactivated fetal calf serum at a final cell
suspension of 3x1 Oslml with O.volvulus parasite antigen (20ug/ml); control
cultures with media only were run simultaneously. The results were expressed as
the stimulation index -SI (counts per minute of stimulated cultures divided by

those of unstimulated cultures).

70

The patients who returned to the clinic with recurrent pruritus
represented less than 10% of the total number of individuals treated in this
particular program. These returnees where categorized according to the
time at which they reported back after their treatment with Mectizan®
(either 0-1 month, 2-3 months, or 4-6 months). They were found to have
significantly higher loads of dermal microfilariae than the control group
(Table 1; Figure 1); eg. 68.4% of pretreatment loads at 4-6 months. Those
who responded to treatment as expected, and did not complain of recurrent
pruritus in this early period after receiving Mectizan®, maintained very low
levels of dermal microfilariae for an extended period of time (rising from
only 1.8% at 1 month to10.1% of pretreatment levels at 4-6 months after

treatment).

There was a reduced proliferative response to Onchocercal antigens in
the early returning groups. The average stimulation index (SI) for the group that
came back after one month was significantly less (2.9+\-0.6) than the control
group (12 normally responding onchocerciasis patients - 12.3+\-1.9; p<0.005).
Those who came after 2-3 months had average SI of 3.2+I- 0.7 and the last

returning group an average SI of 5.6+I-0.8 (Figure 2).

The reason for this reduced ability of Mectizan® to maintain low

levels of dermal microfilariae, and its inability to reduce the major clinical

71

feature of dermal onchocerciasis - pruritus, can only be speculated upon.
All these patients were displaced due to civil unrest and had remained
outside the onchocerciasis endemic zone for more than 15 months, and
thus there was no likelihood of re-infection being the cause of the recurrent
pruritus. Many of them carried onchocercal nodules and all may have had
non-palpable adult containing lesions. Such nodules may serve as sources

of new dermal microfilariae.

The recurrence of pruritus, and apparent resurgence in dermal
microfilariae, may involve a weakening of the paralytic effect of this drug on the
release of uterine microfilariae in the adult worms, or may, as supported by our
findings, reflect an inability of the host’s immune system to contribute to drug-
initiated microfilarial destruction. Such immune mechanisms thus may be less
effective in the patients who show poor parasitological responses to ivermectin,

and lead to the poor clinical response, i.e. the persistence of pruritus.

Our findings differ from previous reports where one annual dose
appeared to be sufficient to curtail microfilarial loads for much longer
periods before any resurgence in parasites occurred (Awadzi et al., 1989;
Brown 8 Neu 1990; Ette etal, 1990; Brieger et al., 1998). This
phenomenon, which although occurring only in a small proportion of
patients, could have a negative effect on general patient participation in a

control program: disappointment with the effectiveness of the treatment

72

could discourage the population at large. It also suggests that, at least in
the treatment of some individuals, Mectizan® may need to be administered
more frequently than once a year as suggested. Such a necessity also
could have important implications to control programs as has been
discussed previously by Richards et al. (2000). Lastly, our observations
support the intriguing possibility that there is an active involvement of the
immune system in the mechanism of action of ivermectin, a concept that

needs further investigation.

73

REFERENCES

Awadzi, K, Dadzie, K. Y., Klager, S. 8 Gilles, H. M. (1989) The
chemotherapy of onchocerciasis. XIII. Studies of ivermectin in
onchocerciasis patients in northern Ghana, a region with long lasting
vector control. Trop. Med. Parasitol. 40, 361-366.

Brieger, W. R., Awedoba, A. K., Eneanya, C. l., Hagan, M., Ogbuagu, K. F,
Okello, D. 0., Ososanya, 0. 0; Ovuga, E. B., Noma, M., Kale, 0. O,
Burnham, G. M. 8 Remme, J. H. (1998) The effects of ivermectin on
onchocercal skin disease and sever Itching: Results of a multicenter
trial. Trop. Med. Internal. Hlth. 3, 951-961.

Brown, K. R. 8 Neu, D. C. (1990) Ivermectin- Clinical trials and treatment
schedules in onchocerciasis. Acta Leiden. 59,169-175.

Boussinessq M. and Gardon J. (1999) La resistance de Onchocerca
volulus a l’ivermectin: une eventualitte a considerer. Annales de
L’Institute Pasteur. 10,81 -91 .

Ette, E.L, Thomas, W, 0. 8 Achumba, J. l. (1990). lvermectin: a long acting
microfilaricidal agent. DCIP. Ann. Pharmacotherap. 24,426-433.

Grant W. (2000) What is the real target for ivermectin resistance selection
in Onchocerca volvulus. Parasitology Today. 16, 458-459.

Richards F, Hopkins D, Cupp E. (2000) Programmatic goals and

approaches to onchocerciasis. Lancet. 355:1663—4.

74

 

Table 3.
Dermal microfilarial loads of the Onchocerciasis Patients Before and After

Treatment with Mectizan®

 

 

 

 

 

 

 

 

 

 

 

 

 

TIME OF POOR POOR POOR NORMAL

OBSERVATION REPONDER RESPONDER RESPONDER RESPONDER

AFTER GROUP 1 ‘ GROUP 21 GROUP 3‘ CONTROL SIGNIF’

TREATMENT (N=16) (N=13) (N=18) GROUP2

(WEEKS) (N=12)

0 113.5 +l- 128.2 +I-10.7 139.9 +I- 11.9 99.1 +I- 21.5 NS

11.9

<4 28.8 +I-3.6 NA‘ NA 1.8 +I-1.1 <0.005

4 - 12 NA 45.8 +I- 6.1 NA 3.6 +I- 2.3 <0.005

13 - 24 NA NA 95.7 +I- 9.7 10.0 +I- 4.1 <0.005
1. Poor responders are grouped into three time periods after treatment depending on

the actual time at which they sought re-treatment for recurrent pruritus.

2. Patients who had the expected response to treatment and whose pruritus had been
continually suppressed since after treatment.

3. Significance of the difierence between the mf loads of the recurrent pmritus and
normal responding groups at each of the time points; Student's t test where NS
(not significant) is > 0.05.

4. NA = not applicable.

75

FIGURE 3.

Recovery of Dermal Microfilariae After Treatment with Mectizan®.

% of Pretreatment Dermal Microfilarial Load

 

 

E'IPRURITIC '
RESPONDERS

.. ; INORMAL

' RESPONDERS

 

 

 

 

 

 

0 - 1 MONTH 2 - 3 MONTHS 4 — 6 MONTHS
TIME, OF REPRESENTATION AT CLINIC

 

 

 

 

76

FIGURE 4.

Lymphocfle Transformation in Response to Culture with Onchocerca
volvulus Specific Antigen.

 

% SI of

Control

Patients
Cells

 

 

 

 

 

PATIENT GROUPS'

 

a. Patient groups

1. Control patients

2 Those retuming in less than I month

3. Those returning at 2-3 months after treatment
4

Those returning at 4-6 months after treatment

77

Summary
Both Cell Mediated Immunity (CMI) and Antibody Mediated Immunity (AMI)
are involved in the development Of dermal pathology in onchocerciasis
There is a positive relationship between cell cytO-adherence, active cell
mediated immunity, microfilarial loads and the severity of dermal disease.
Cellular immune responses to the parasite antigen appeared to mirror those
of humoral responses.
Treatment with ivermectin (MectizanR) intimately involves the immune system,
either:
As an "after effect” as the case of patients who their immune
responses were being enhanced after treatment, or is involved in
the drug’s action as suggested for the returnee group of patients.
lmmunologically compromised individuals may not respond
efficiently in treatment.
The importance Of including clinical evaluation in the epidemiological
assessments Of disease prevalence rather than relying only on parasitological
parameters.
The mass drug administration programs should not exclude hypoendemic
areas in order not to miss areas where people suffer the most from the severe

onchodermatitis.

78

 

Figure 5.

 

 

PARASITE (Strain) HOST (Genetics) DRUG EFFECTS
(Antigen) (History) (lvermectin, DEC)

 

 

 

 

 

 

 

 

\ ML... /

SUPPRESSION C T > REACTION
IIIIHCIIRHMIHII @El‘i'lilii]

 

 

 

 

 

 

 

 

 

 

 

Mf DESTRUCTION NO Mf DESTRUCTION

 

 

 

 

 

 

 

 

CLINICAL RESPONSE LESS CLINICAL RESPONSE

 

 

 

79