DETECTION OF MALARIA ANTIBODIES USING PLASMODIUM KNOWLESI

ANTIGEN

Dissertation Submitted for the Degree of

Mdiittv of $l)ilo!B;opI)p

MICRDBIDLDGY ' ^ l ^ r' TO ' -̂ ^5te JO j II I ^ THE FACULTY OF MEDICINE

' ALIGARH MUSLIM UNIVERSITY ALIGARH

FARRAH MIRZA

Approved :-

Prof. SOHAIL AHMAD Supervisor

PARASITIC IMMUNOLOGY LABORATORY DEPARTMENT OF MICROBIOLOGY

JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MVSLIM UNIVERSITY

ALIGARH (INDIA)

1 9 9 2

2 7 JUN 1994

DS2329

•• ; ii^

DEDICATED

TO

MUMMY. DADDY AND GUDIYA: WITH YOU ALL HERE

'HEAVEN IS A PLACE ON EARTH'

PARASITIC IMMUNOLOGY LABORATORY DEPARTMENT OF MICROBIOLOGY

JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MUSLIM UNIVERSITY

ALIGARH

CERTIFICATE

This is to certify that the work presented in this

dissertation was carried out by Ms. Farrah Mirza in my

laboratory. It is suitable for the award of the degree of

Master of Philosophy fM.Phil) in Microbiology.

(Prof.Sohail Ahmad) M.S. (U.S.A.), Ph.D.

LIST OF CONTENTS

INDEX PAGE

LIST OF FIGURES i

LIST OF TABLES ii

ACKNOWLEDGEMENTS iii

ABSTRACT vi

INTRODUCTION 1

1. Historical Review 2 2. Classification 3 3. Malaria Life Cycle 4 4. Immunity to Malaria 6 5. Malaria vaccines 18 6. Plasmodium knowlesi - The simian malaria

parasite 24

AIMS AND OBJECTIVES 26

MATERIALS 28

METHODS

- Maintenance of Plasmodium knowlesi cell 30 1 ine

- Counting of Parasites 30 - Staining and Fixation of Blood Films 31 - Antigen Isolation 31 - Preparation of Water Soluble Whole Antigen 32 - Protein Estimation 32 - Measurement of pH 32 - Indirect Haemagglutination Test 32 - Treatment of Infected Animals 34

RESULTS 35

DISCUSSION AND CONCLUSION 47

BIBLIOGRAPHY 55

LIST OF FIGURES

FIGURE 1 : Seropositivity of Malaria Specific antibodies by the IHA tests in the Blood film positive and Blood film negative cases

PAGE

38

FIGURE 2 : Photograph of Indirect Haemaggluti- 41 nation test

FIGURE 3 : Photomicrograph of a blood smear 42 from an infected monkey showing schizout stage of the parasite

FIGURE 4 Distribution of P. vivax and 44 P. falciparum in Blood film positive malaria patients

FIGURE 5 : Frequency of IHA titres in malaria 46 cases and controls

11

LIST OF TABLES

PAGE

TABLE I Seropositivity of malaria antibodies in clinically diagnosed malaria patients using indirect haemaggluti-nation test (IHA)

37

TABLE II : Seropositivity of malaria antibodies in normal control patients using IHA test

39

TABLE III : Sex distribution of malaria patients

side positive 40

TABLE IV : Distribution of Plasmodium vivax and Plasmodium falciparum in s1ide positive malaria patients

43

TABLE V Distribution of patients

IHA titres in malaria 45

ill

ACKNOWLEDGEMENTS

With the passage of time things often take on a dream­

like quality. Thus, today, nearly two years after first

Joining this department for my M.Phil, my memories of the

first few days are a bit hazy. All I seem to remember is the

nervousness which I think didn't have much to do with the

change of an entire continent (having done my M.Sc. from

Nigeria) but was more due to the apprehension of having to

meet my new Supervisor, Dr. Sohail Ahmad.

However, now I can sit back and remember things with a

smile, for my M.Phil stands completed and who can I thank

more than Dr. Sohail Ahmad who was always there to push me

forward; it was his words of encouragement and his confidence

in my abi1ities which saw me through the 1abyrinth of

research work which needs to be done before one reaches the

place where I am today - very near a M.Phil degree. I Just

can't find words to thank him for everything, all I can say

is that, to me this laboratory and department would somehow

fail to exist without this pillar of strength.

I must also thank Dr.Ashok Bal who now captains the

ship of the department of Microbiology and makes sure that it

overcomes all tides that pass its way.

IV

The mouthwatering doughnuts, fabulous biryanis, all the

other wonderful delicacies and above all the kindness and

encouraging smile make it extra hard for me to forget one

person - Mrs Sohail Ahmad. Ma'am, you have really made this

laboratory 'A home away from home'.

How can I forget Dr.Maris Manzoor Khan who always lent

a patient ear to all my problems and managed to find somewhat

ingenious solutions for them. His sense of humour was the

buoy that kept me from drowning despite the numerous tides

that I had to tackle.

Hemant Bhai, without you who would I have gone to for

all that useful information and those wonderful suggestions?

Not to forget - the markers, the samosas and numerous cups of

'Chai'.

Thanks - Raka di, Ghazzanfar bhai, Khalid bhai. Sabiha,

Azra, Huma, Ritu, Chandrama, Dr.MukerJee and Zakir for being

the 'Wind beneath my wings'.

Munna Lai and BriJ Mohan despite being most helpful

will welcome the new season with a sigh of relief because

with my M.Phil complete there will definitely be a decline in

their busy 'errand' schedule.

Special thanks are due to the people at the Animal

house because without them I might have been 'caged' - with

the monkeys!

Mr. Suhi: i Is meticulous typing lids worked wonders for the

few pages whicn have taken on a new shape - that of a

presentable thesis.

Zafar Eqbal was ever ready to help even though mostly

he ended up running around in circles. However, as they say

'Its the thought that counts! ' Thank you.

Arif, but for you, I'd still oe browsing through my

papers without going forward even a stop. You have

definitely proved that you are the silver lining in Al igarn ' s

c;-Or,'a, Oops ! I me An t c !oud.

VI

ABSTRACT

PIasmodi urn know!es 1 cell line was maintained and

allowed to multiply, in vivo, in Rhesus monkeys. Daily blood

films were made to observe the appearance of parasites in the

peripheral blood. The parasitaemia reached to about 80^ on

the 12th day of infection.

The parasites were isolated through the use of numerous

centrifugations, washings and lysis of the erythrocytes by

the use of saponin. The free parasites were disrupted by

ultrasonication and the suspension was centrifuged again to

obtain the antigen.

Blood films and sera specimens were taken from 50

patients with clinically diagnosed malaria from the J.N.M.C.

Hospital Out Patient Department and from those who were

admitted to the J.N. Medical College Hospital wards, Aligarh,

India.

The blood films were stained by Leishman stain and

observed under the microscope for the presence of malaria

parasites. Indirect haemagglutination tests were done on the

sera samples to determine malaria parasite specific antibody

titres against P. knowlesi antigen.

VI1

The parasitologic examination of slides yielded 21

(42%) blood films positive for malaria parasite and 29 (58%)

negative blood films. The sera of patients with positive

blood films gave a positive antibody titre (_>1:16) in &)%

cases, while the sera from patients with negative blood films

were about 73* IHA positive.

26 control patients were also included in the study

(that is those with no clinical evidence of malaria and

showing a negative blood film) from whom blood films and sera

specimens were taken. Only 1 (4%) serum sample gave a

positive titre in the above control groups.

INTRODUCTION

Malaria is a communicable disease caused by Sporozoon

parasites of the genus Plasmodium and transmitted to man by

certain species of infected female Anopheline mosquitoes.

The disease is characterised clinically by 3 stages.

(a) Cold stage: sudden onset of fever with rigor and

sensation of extreme cold accompanied by shivering.

(b) Hot stage: patient feels burning hot and casts off

all his clothes, there is intense headache.

(c) Sweating stage: fever comes down with profuse

sweating. There is enlargement of spleen and

secondary anaemia with a tendency to relapses (WHO,

1986) .

Malaria is a threat to almost half the world's

population. It is a major health problem in tropical

countries, where approximately three hundred million cases

occur annually. In Africa alone, almost one to two million

children die of the disease every year (Siddiqui, 1991). In

India an alarming increase in malaria cases has been

reported since 1965 and the maximum number has been

recorded in 1976 (6.5 x 10^) (Ghatak ^^ al-, 1987). The

number of confirmed cases recorded in 1988 were 1.78

million (WHO, 1990).

2 HISTORICAL REVIEW

Malaria is caused by a protozoan parasite of the

genus Plasmodium. The first accurate clinical descriptions

of malarial fevers were given by Hippocrates in 400 B.C.;

(Boyd, 1949) who mentioned the classic triad of chills,

fever and sweating. It seems that malarial fevers were

also known in ancient China and that Arab physicians during

the eight to the thirteenth century were commonly

acquainted with intermittent fevers (Boyd, 1949).

The first treatment of malaria came in the middle of

the seventeenth century with the introduction of the bark

of a Peruvian tree which was subsequently called cinchona.

Pelletier and Caventon, in 1820, succeeded in extracting

quinine from cinchona which was found to have an

antimalarial effect (Scott, 1939).

Meckel, in 1847, showed black granules, embedded in

protoplasmic masses, in the blood of patients suffering

from malaria. In 1880, Laveran, a French Army surgeon

discovered the malaria parasite in Algiers, North Africa.

In 1891, Romanowsky in Russia developed a new method of

staining blood films which made possible complete studies

of the malaria parasite. It was proved by Gerhardt (1884)

that malaria could be induced in healthy persons by

3

inoculation of blood of severely ill malarious patients.

Manson (1894) hypothesised that mosquitoes transmitted

malaria and Ronald Ross (1897) found malaria parasite of

man, growing as cysts (oocysts) on the stomach wall of an

Anopheline mosquito which had previously fed on a malaria

patient.

CLASSIFICATION

There is still a lot of discussion and uncertainty

about the classification of malaria parasites. The

classification of the phylum protozoa introduced by the

society of Protozoo1ogists (Honigberg gjfc. al- , 1964)

provides a generally accepted system based on morphological

and biological criteria, as well as on phylogenetic

considerations. According to this classification, the

malaria parasites belong to the subphylum Sporozoa, class

Telesporea and subclass Coccidia.

All members of the Subphylum Sporozoa are parasitic

of which some produce resistant spores or have a stage in

their life cycle which is derived from a resistant spore;

these forms contain one or more sporozoites. All

Telosporea possess sporozoites and reproduce sexually. The

common characteristic of subclass Coccidia is the

intracellular nature of the trophozoites.

MALARIA LIFE CYCLE

More than 100 plasmodial species have been described,

causing malaria in a wide range of vertebrates and

exhibiting narrowly defined host specificity. Four species

infect man, P. falciparum. £. vivax. P, malaniafi, and P.

ovale, the first being the most lethal. P. vivax has the

widest distribution throughout the world. In India, 65 to

69 per cent of the infections are reported to be due to P.

vivax. 25 to 30 per cent due to P. falciparum and 4 to 8

per cent due to mixed infections. P. malariae is said to be

responsible for less than one per cent of infections in

India (Park and Park, 1386).

The malaria parasite undergoes two cycles of

development, the human or asexual cycle and the mosquito or

the sexual cycle. Man is the intermediate host and mosquito

the definitive host.

Asexual Cycle: The asexual cycle begins when an infected

mosquito bites a person and injects sporozoites. Four

phases are described in the human cycle.

(a) Pre-erythrocytic phase: The sporozoites disappear with

half an hour from peripheral circulation and reach the

liver cells, where they undergo a cycle of development

which lasts for 6 to 8 days. At the termination of this

5

phase a shower of parasites (cryptozoites) are liberated

into the blood circulation (Garnham, 1966; Ayala, 1977).

(b) Erythrocytic Phase: The parasite passes through the

stages of trophozoite and schizont. The phase ends with

the liberation of merozoites which infect fresh red cells.

This release of merozoites into the blood stream causes

fever, the prime symptom of malaria.

(c) Gametogony: Some of the merozoites penetrate the red

cells but instead of developing into schizonts, they become

gametocytes. These are the sexual forms of the parasite

which are infective to mosquito (Aikawa gj;: ,aJ.. , 1978).

(d) Exoerythrocyti c Phase: In £. f_aJJLiP_aJliLDl the

intrahepatic schizonts rupture almost simultaneously and

there is no persistent tissue phase. The intrahepatic

schizonts of other Plasmodia do not burst all at the same

time and may remain dormant for an indeterminate period.

The cycle remains potentially active for 1 to 3 years for

£• vi vax and P. ovale. In P. malari ae recurrences are

possible 10, 2 0 and even 40 years after sporozoite

inoculation. The exoerythrocytic cycle is sustained

exclusively by blood schizogony or released from liver,

cells are not capable of reinfecting the liver (Park and

Park, 1986).

Sexual Cycle: The sexual cycle or sporogony is initiated

when an Anopheline mosquito capable of transmitting malaria

feeds on a malaria patient. In the lumen of the midgut,

the gametocytes escape from the host cells and soon undergo

changes preparatory to fertilization. The male gametocyte

(microgametocyte) through a process known as

"exf1agellation" form a number of sperm-like bodies.

Fertilization occurs when a microgamete- enters a

macrogamete by chemotaxis and a zygote is formed. The

motile zygote or ookinate penetrates the stomach wall of

the mosquito and after about 24 hours develops into an

oocyst on the outer surface of the stomach (Sinden, 1975;

Speer £i M-, 1975). The oocyst grows rapidly and develops

within its numerous sporozoites. The oocyst ruptures into

the body cavity after about 2 weeks and many of the

sporozoites eventually find their way into the salivary

glands of the mosquito which now become infective to man

(Joklik fii al., 1984).

IMMUNITY TO MALARIA

Innate resistance influences the pattern of acquired

malarial immunity which differs widely in distinct host-

parasite combinations and even with the same parasite in

closely related host species. The mechanisms of innate

resistance has been shown to influence the development of

7

a l l asexual blood forms but none of those res is tances seem

to act upon gametocytes ( M i l l e r and Car te r , 1976).

I n d i v i d u a l s t h a t lack the Duffy b lood-group ant igens

on t h e i r e r y th rocy tes are r e s i s t a n t t o P. v ivax i n f e c t i o n ,

because the receptor f o r the P. v ivax merozoi tes on the red

c e l l i s assoc ia ted w i t h ant igens of the Duffy blood group

(Butcher ^ a l - , 1973; M i l l e r e i a l . , 1973, 1978; Barnwell

e t a l . • 1 9 8 9 ) . T h i s e x p l a i n s t h e l o n g r e c o g n i s e d

res is tance t o P. v ivax i n f e c t i o n of West A f r i c a n blacks and

t h e i r descendants i n the USA. Because these popu la t ions

have a h igh f requency o f the D u f f y n e g a t i v e genotype

( M i l l e r e i a_L- , 1 9 7 6 ) . H e t e r o z y g o t e s f o r

haemoglobinopathies and other d i so rders of the red c e l l s

( t h a l a s s e m i a and g 1 u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e

d e f i c i e n c y ) promote i n n a t e r e s i s t a n c e t o P.. f a l e i oarum

i n f e c t i o n (Wea the ra l l , 1987). Such p r o t e c t i o n i s probably

t r i gge red by m o d i f i c a t i o n s of p a r a s i t e development w i t h i n

the e r y t h r o c y t e s o f these i n d i v i d u a l s . However, i t has

been proposed t h a t these simple p h y s i o l o g i c a l e f f e c t s may

f o s t e r p r o c e s s e s t h a t l e a d t o an enhancement i n t h e

i n t e n s i t y a n d / o r s p e c i f i c i t y o f t h e a d a p t i v e immune

response, e n a b l i n g i n d i v i d u a l s c a r r y i n g t h e s e genes t o

acqu i r e c l i n i c a l immunity t o m a l a r i a f a s t e r t h a n o t h e r s

( A l l i s o n , 1984 , B a y o u m i , 1 9 8 7 ) . I n s u p p o r t o f t h i s

8

hypothesis Bayoumi sX. al (1990) found that lymphocytes of

individuals carrying the HbAS genotype showed higher

reactivity to malaria antigens in proliferative assay than

lymphocytes from HbAA controls living in the same area.

Protection to malaria, acquired by individuals living

in endemic areas is largely governed by the transmission

pattern. MacDonald (1957) identified two epidemiological

extremes in which the disease exists, one where instability

of transmission is the dominant feature and the disease is

characterized by substantial seasonal fluctuations,

producing cyclical epidemics in the entire population

regardless of age. In the other case, transmission and

morbidity remain stable over the seasons and years, with

the population exposed to frequent heavy inoculations. The

individuals appear to acquire protection against clinical

attacks after several years (Theander, 1992) and the

disease therefore affects mainly children.

McGregor (1960) proposed that individuals living in

areas with stable malaria pass through five stages before

immunity is acquired. In stage one (age group 0-2 months)

infants are protected against malaria and parasites are

only found in the peripheral blood of approximately 10*

(Gilles, 1961).

9

During stage two (2-6 months) parasites are found

more frequently in the blood (Gilles, 1961) and the

children often experience the first mild clinical attack of

malaria.

Stage three (7 months-2 years) is characterized by

repeated attacks of malaria which are associated with

severe illness. Parasites are almost always found in the

peripheral blood in high densities. Malaria disrupts the

normal pattern of growth in this age, producing severe

anaemia and high mortality rate (McGregor gi aJ_. , 1956).

Stage four (3-4 years) represents the acquisition of

the first phase of clinical immunity characterized by a

rapid decline in the severity and.frequency of clinical

episodes, despite the persistence of a relatively dense

parasitaemia.

In the fifth stage clinical malaria attacks are rare

and mild. Adults living in the area often harbour the

parasite without clinical symptoms. Reports by Colbourne

(1955) on susceptibility to malaria in students returning

to Africa after studies in Europe, and by Mastbaum and

Goeckel (as quoted by Bruce-Chawatt, 1963) on malaria

outbreaks suggest that immunity diminishes in individuals

living in areas free of transmission. Whether this is due

10

t o i n t r o d u c t i o n of new p a r a s i t e s t r a i n s when exposure was

r e s t o r e d or due t o lack o f i m m u n o l o g i c a l memory i s no t

known.

Thus i n d i v i d u a l s l i v i n g in areas of s t ab l e malar ia

t ransmiss ion develop p r o t e c t i o n to the devas ta t ing e f f e c t s

of the d isease over a number o f y e a r s . Such p r o t e c t e d

i n d i v i d u a l s are c h a r a c t e r i z e d by t h e r a r i t y o f c l i n i c a l

symptoms and t h e i r mi ldness i f an a t tack of ma la r ia occurs .

Acquired immunity i s on ly p a r t i a l , as pa ras i t es are found

i n low d e n s i t y i n t h e p e r i p h e r a l b l o o d o f p r o t e c t e d

i n d i v i d u a l s and immunity wanes once t h e exposu re t o t h e

pa ras i t e ceases.

Defence mechanisms o f immun i ty a re med ia ted and

regu la ted through an i n t e r p l a y between d i f f e r e n t pa r ts of

the system. The c l a s s i c a l d i v i s i o n i n t o the s p e c i f i c and

non -spec i f i c pa r ts of the system and between B and T - c e l l

m e d i a t e d i m m u n i t y becomes l e s s d i s t i n c t when i t i s

understood t h a t T - c e l l s f o r instance s t i m u l a t e non -spec i f i c

N a t u r a l K i l l e r (NK) c e l l s and m o n o c y t e s t h r o u g h t h e

s e c r e t i o n o f I n t e r l e u k i n -2 - f I L - 2 ) and i n t e r f eron- lJ

( IFN->^ ). T - c e l l s are on l y a c t i v a t e d when exposed t o

a n t i g e n by a n t i g e n p r e s e n t i n g c e l l s . The e f f e c t o f

an t ibod ies are o f t en t o opsonize micro-organisms so t ha t

11

they are easily phagocytized, the T-cells regulate the

production of antibodies by B-cells. (Theander, 1992).

The premise that macrophages, or their products are

important for protection is supported by experiments in

which mice were protected against sporozoite-induced

(Nussenzweig, 1967) or blood-stage induced malaria

infections CClark ei al., 1976, 1977) by non-specific

stimulation of the phagocytic defence systems through the

administration of BCG. Cells of the macrophage lineage and

neutrophils are also able to kill liver stages of the

parasites and have been shown to infiltrate liver tissue

and destroy rat hepatocytes infected with P. berqhei (Meis

gLl a,I. , 1987).

Monocytes or macrophages eliminate malaria parasites

both by phagocytosis and through secretion of molecules

toxic to the parasite. Peripheral blood monocytes from

malaria unsensitized donors are able to attach to and

ingest P. falciparjum merozoites. Phagocytosis is markedly

enhanced in the presence of immune serum (Krusmith and

Druilhe, 1983). Results obtained in a mouse model indicate

that non-activated macrophages primarily bind Plasmodia via

receptors for carbohydrate containing glycoproteins, while

activated macrophages bind them via Fc receptors (Brown and

Kreier, 1986). Phagocytosis seems to be regulated by the

12

T-cells as Plasmodium antinen-stimu1ated T-cel1s from

spleens of malaria infected mice produced a lymphokine

capable of activating macrophages for enhanced Fc-receptor

mediated phagocytosis (Ockenhouse and Shear, 1983). Malaria

parasites trigger macrophages to release parasite toxic

mediators and the activity is regulated by T-cells

(Ockenhouse and Shear. 1984).

Macrophages play an important role as antigen

presenting cells. T-cell receptors co-recognize complexes

of antigen and products of major histocompatibility complex

(MHO on the surface of other cells. Subpopulations of

antigen presenting cells handle antigens differently, and

the way monocytes process and present antigens play a role

in activation of subpopulations of T-cells, whereby they

influence the outcome of the activation process (Theander.

1992).

Besides their role in protection, the macrophages

appear to play a role in the pathogenesis of the disease.

Malaria antigen can stimulate tumour necrosis factory:

(TNF- oC ) and IL-1 production in macrophages (Jakobsen e_t

al-, 1990), although these can induce anti-parasitic

effects. They probably also contribute to the symptoms arid

complications of malaria (Clark ejt a±. . 1989). Both

13

monokines can induce fever and they seem to play a critical

role in the pathogenesis of cerebral malaria (Grau et al..

1987).

Neutrophils can inhibit the growth of P. falciparum

in vitro (Brown and Smalley, 1981; Nnalue and Friedman,

1988). They probably kill the parasite and have been shown

to phagocytize parasitized erythrocytes j_n vivo (Kharazmi

e.t al. , 1987 ) and in vitro (Celada et al- . 1983).

Little is known about the role of eosinophils in the

protection against malaria as the infection does not result

in eosinophi1ia. The in vitro growth of P. falciparum can

be inhibited by granule proteins secreted by purified

eosinophils. Both the eosinophil cationic protein and

other granule products seem to be toxic to parasites as

monoclonal antibody against the cationic protein only

partially blocked the inhibitory effect (Waters et a]..

1987) .

T-cells might participate in the protection against

malaria as (1) helper cells for antibody production, (2)

through the activation of non-specific effector cells like

macrophages or granulocytes, (3) through the production of

substances toxic to the parasites and (4) as cytotoxic

cells (Theander, 1992).

u A l t h o u g h T and B - c e l l s can c o n v e y some i m m u n i t y ,

o p t i m a l p r o t e c t i o n i s o b t a i n e d by t h e t r a n s f e r o f T - c e l l s

o f t h e Ly-1 pheno type , i n c o m b i n a t i o n w i t h B - c e l l s .

A l t h o u g h T and B - c e l l s can c o n v e y some i m m u n i t y

o p t i m a l p r o t e c t i o n i s o b t a i n e d by t h e t r a n s f e r o f T - c e l l s

o f t h e L y - I pheno type , i n c o m b i n a t i o n w i t h B - c e l l s .

However, T - c e l l s a re a l s o ab le t o convey immuni ty i n

a n t i b o d y i n d e p e n d e n t s y s t e m s . B - c e l l d e f i c i e n t m i c e

t e r m i n a t e t h e i r i n f e c t i o n i n t h e same way as

i m m u n o l o g i c a l l y i n t a c t m i c e , t h e p r o t e c t i o n depends on the

presence o f an i n t a c t s p l e e n (Grun et al. , 1985) . T - c e l l s

m i g h t a l s o p l a y a r o l e i n p r e v e n t i n g t r a n s m i s s i o n o f

m a l a r i a as H a r t e e i a j_ . ( 1 9 8 5 ) w e r e a b l e t o r e d u c e

t r a n s m i s s i o n by 95% by p a s s i v e t r a n s f e r o f T - c e l l s f r o m

immune t o u n p r o t e c t e d a n i m a l s .

S e r o - e p i d e m i o l o g i c a l s t u d i e s o f t o t a l m a l a r i a

s p e c i f i c a n t i b o d i e s have i n d i c a t e d t h a t t h e r e i s an age

dependent i n c r e a s e i n a n t i b o d y l e v e l s i n i n d i v i d u a l s l i v i n g

i n m a l a r i a endemic a r e a s ; t h e l e v e l o f t hese a n t i b o d i e s i s

not an index o f f u n c t i o n m a l a r i a immun i t y , bu t an i n d i c a t o r

o f t h e d e g r e e o f m a l a r i a e x p o s u r e i n t h e p o p u l a t i o n

( V o l l e r , 1 9 7 1 ) . The a n t i b o d i e s a r e d i r e c t e d a g a i n s t

s p o r o z o i t e ( N a r d i n e t a l . , 1979) as w e l l as b lood s tage

15

(Cohen and Butcher. 1971) and gametocyte (Mendis et a1..

1987) antigens.

Complement does not seem to be able to kill parasites

directly, but could play a role as an opsonin for

neutrophils and macrophages (Salmon gt al., 1986).

The acquisition of immunity is a slow process during

which the individual is exposed to the parasite on numerous

occasions. The parasite could (1) prevent or divert the

immune recognition and (2) inhibit the immune responses to

antigens that have, or could be recognized by the immune

system. During blood stage infections, the parasite is

mostly intracellular and exposed to the extracellular

environment only for short periods of time before it

invades a new host cell. The parasite is partly hidden

from the immune system. The erythrocytes infected with

late trophozoites and schizonts bind to the endothelial

cells of the capillary system, thus avoiding the effector

cells of the spleen and other tissues.

More than twenty P. f alci parum proteins have been

shown to share amino acid sequences with human proteins

(McLaughlin gt al., 1987). The blood stage antigen Pf 11.1

shows homology with a thymic peptide (Dubois et al., 1988).

a gametic stage antigen contains segments homologous with

16

epidermal growth f a c t o r (Kaslow et_ a j . . , 1988) and the

cen t ra l repeat segment of the c i rcumsporozo i te p r o t e i n is

homologous w i t h a m i tochondr ia l p r o t e i n (McLaughlin e t a l . ,

1987). The e f f e c t of these homo log ies m i g h t be t h a t

o the rw ise immunogenic segments o f m a l a r i a p r o t e i n s are

recognized as ' S e l f and t o l e r a t e d by the immune system.

Paras i tes have been repor ted to cross the p lacenta and gain

access t o the f e t a l c i r c u l a t i o n b e f o r e and d u r i n g b i r t h

(Reinhardt e t al. , 1978). Although these c h i l d r e n seldom

develop c l i n i c a l ma lar ia (McGregor, 1984), to le rance might

be induced f o l l o w i n g the prenata l or p e r i n a t a l exposure to

malar ia ant igens (Pombo e t aj_-, 1988).

The pa ras i t es appear t o be able to undergo an t igen ic

v a r i a t i o n w i t h i n a s i n g l e c l o n e and exp ress a l t e r n a t i v e

forms of a molecule from a given genie r e p e r t o i r e (Hommel

e t al . . 1983, Fenton ejt a j_- , 1985 ) . Such d i v e r s i t y i n

m a l a r i a p a r a s i t e s i s l i k e l y t o c o n t r i b u t e t o the s low

development of immuni ty . Many m a l a r i a p r o t e i n s c o n t a i n

shor t amino ac id sequences t h a t are ex tens i ve l y repeated in

tandem a r r a y s . These r e p e a t e d sequences encode

immunodominant e p i t o p e s t o wh ich much o f t he a n t i b o d y

response a g a i n s t the p a r a s i t e i s d i r e c t e d (Kemp eX. a l . ,

1987). An t ibod ies aga ins t one such ep i tope o f t en c ross-

react w i t h o ther repeated ep i topes , e i t h e r w i t h i n the same

17

molecule or on other parasite proteins. Anders (1986)

proposed that these cross-reactions interfere with the

maturation of hiqn affinity antibody directed against the

parasi te.

Acute malaria infection causes marked perturbations

in the immune system of the host. On one hand, the

infection induces a powerful polyclonal activation of the

immune system resulting in hyper-gamma globulinaemia and

production of auto-antibodies, and on the other hand the

infection results in a depression of immune reactivity.

Symptoms and signs of respiratory and gastrointestinal

infections are common in children with acute malaria, but

this may be attributed directly to the parasite (Greenwood

ejt a.!., 1989). Clinical reports have indicated that

Salmonella infections are especially severe in malarious

children (Bennett and Hook, 1959). Although clinical

evidence for immuno suppression during malaria is not

overwhelming, results of these studies on P.. falciparum

malaria plus convincing evidence showing immunosuppression

during malaria in animals has led to a series of studies in

which immune reactivity during P. falciparum malaria was

characterized jm vi tro (Weidanz, ^962].

18 MALARIA VACCINES

Accordinq to WHO, about 100 million people are

clinically ill with malaria at any given time, and around 1

million - mostly very young children in Africa - die from

it each year. The effects are so widespread in the tropics

- ranging from lethargy to total incapacitation - that

malaria has been blamed for impeding the development of

entire nations and the problems with malaria will be

getting worse in the years ahead.

The main strategy for fighting malaria through the

1960s was to use pesticides to kill mosquitoes that carry

the parasite and to treat infected individuals with quinine

- related drugs, mainly chloroquine. However, the most

lethal strain of the parasite, P.. falciparum is now

genetically resistant to chloroquine. Health authorities

are switching to a new compound, mefloquine, but already

there are signs in Asia and Africa that the parasite is

resistant to this as well. It is only a matter of time

before the resistance spreads (Cherfas, 1990).

Malaria vaccines will be based on pure parasite

antigens which specifically stimulate protective immune

responses. Protective antigens are present in several of

the developmental forms of the parasite and future vaccines

19

may contain antigens from one or more of these, at present

most of the relevant research concerns P.. f a1 c i parum.

Vaccine trials currently envisaged are: (i) Sporozoites,

(ii) asexual erythrocytic stage, and (iii) gametes and

other forms developing in the mosquito midgut (WHO, 1986).

S-antigens of P. falci parum have also been successfully

employed for vaccination experiments (Ristic et al., 1985).

Vaccination of rodents, monkeys, birds and humans

with X-irradiated sporozoites induces protective immunity

to subsequent challenge with living sporozoites (Strickland

and Nussenzweig, 1982). Preliminary attempts at sporozoite

vaccination were made in 1971 - 1975 using X-irradiated

sporozoites of P. f al ci parum and P_. v i v a x which were

innoculated by mosquito bites into previously non-exposed

human volunteers. This bold trial brought to light the

information that the immunity to the Sporozoite challenge

became detectable 7-10 days after innoculation, immunity

was species and stage specific and appeared after 10 days

of immunization and persisted for 3-5 months and, no side

effects to the vaccine were observed (WHO, 1986). The most

promising achievement with regard to the development of a

sporozoite vaccine was gene cloning of P. knowlesi antinens

in Escherichi a coli. This was done by extracting the

messenger RNA encoding circumsporozoite protein from

20

infected mosquitoes through conversion, by enzyme reverse

transcriptase, into CDNA. The fragments of CDNA were

inserted into bacterial plasmids which were in turn

introduced into £. coli. The bacterial clones excreted

peptides binding to the monoclonal antibodies and against

circumsporozoite protein of P. knowlesi (Sharma and Godson,

1985) .

Sequencing analysis of circumsporozoite protein

showed that the immunodominant antigenic determinant

(epitope) contains a sequence of 12 amino acids and the

peptides share common antigens with the original

circumsporozoite protein (WHO, 1985).

A sporozoite vaccine, if fully effective, would

prevent the establishment of plasmodial development in the

host and thus induce sterile immunity. Parasitaemia,

asexual or sexual, would therefore not occur; clinical

illness would not supervene and the subject would remain

incapable of infecting mosquitoes. A vaccine of this type

should be able effectively to interrupt the natural

transmission of malaria; however, subjects effectively

immunized against sporozoites will remain susceptible to

challenge with asexual erythrocytic stages of the parasite

(WHO. 1986).

21

The merozoite or an asexual erythrocytic-stage

vaccine is expected to induce an immunity that operates by

restricting the replication of asexual blood stage

parasites without necessarily inducing sterile immunity.

Consequently its function will be to reduce the morbidity

and mortality due to malaria. However, persons immunized

by this type of vaccine will remain susceptible to

sporozoite infection and the parasites development in the

liver will occur unimpeded; low grade asexual parasitaemia

may occur and gametocytogenesis may evolve normally so that

immunized persons would remain capable of infecting

mosquitoes. It is unlikely that the asexual blood stage

vaccines if used alone will achieve the interruption of

malaria transmission in any endemic area; however they can

be used in highly susceptible groups in endemic areas to

induce a level of immunity which prevents serious illness

after infection. The merozoite vaccine acts as a curative

measure, and will overcome the problem of drug resistance.

Irradiated parasitized erythrocytes have been extensively

utilized as experimental immunogens. Some success was

achieved with rodent malaria (Wellde and Sadun, 1967) in

which irradiated erythrocytes infected with P. faJclEAriM

were used to vaccinate owl monkeys (Wellde et a_i. . 1979).

Two significant findings of these studies show that a

degree of protection could be obtained without using an

22

adjuvant; and that four immunizinq doses were required to

achieve significant degree of immunity.

The third type of vaccine will operate by inducing in

the human host serum antibodies that effectively block the

fertilization of female gamete by male gametes v*ithin the

mosquito gut, or inactivate the fertilized zygote or the

ookinete. Such transmission blocking vaccines will

apparently be capable of interrupting malaria transmission

at the mosquito level, but will neither protect the human

host against sporozoites, asexual blood stages, nor prevent

the development of gametocytaemia. However, they are

expected to reduce the overall rate of malaria transmission

in endemic areas and thereby play an important role in

control (WHO, 1986). The major prerequisite for producing

a gamete vaccine is the availability of large number of

infectious P. falci parum gametes and the continuous in

vitro cultivation of P. falciparum has given great impetus

for achieving this goal.

The S-antigens of P. falciparum are the subject of

great deal of research for vaccine production. Th.ey incucf

significant protective immunity in Squirrel monkeys against.

a virulent challenge by P. falciparum (Ristic e_t aj. • •

1985). Shamansky e_t a_l. (1985) have purified two S-

23

antigens (83 and 100 KDa) and have proposed that these may

be good candidates for potential host protective antigens

in malarial immunoprophylactic regimens.

Development of successful malaria vaccines will

depend on both the production of protective antigens by

chemical synthesis or recombinant technology as well as the

availability of safe and effective adjuvants. The best

protection has been achieved only when monkeys were

immunized in conjunction with Freund's complete adjuvant

(FCA).

Results of clinical trials with sporozoite and

merozoite vaccines with alum as an adjuvant have shown

limited immunogenicity and protective success. Since FCA

is unacceptable for clinical use, as it has toxic side

effects, and results of clinical trials with alum have

shown limited success, as it shows humoral response but is

deficient in inducing a ce11-mediated response; several

laboratories have sought to establish a safe and effective

adjuvant. Saponin has been found to be an effective

adjuvant with rodent malaria antigen but its efficacy with

purified or subunit human malaria antigens has yet to be

studied. Based on knowledge generated on the immunology of

malaria, it is evident that a malaria vaccine adjuvant must

24

be capable of inducing both humoral and cell mediated

responses (Siddiqui, 1991a).

The prospect for the development of effective malaria

vaccines is reasonably good, but the realisation of this

goal will require some time. Parasite antigenic diversity

and genetic restrictions of immunological responses to

these antigens in the human population pose great problems

to the effectiveness of subunit malaria vaccines now beinn

developed.

Plasmodium knowlesi - THE SIMIAN MALARIA PARASITE

The parasite is remarkable for its unique quotidian

periodicity in the blood and it is almost 10051̂ lethal in M.

malatta: however in M. fasciculari s it is not lethal as the

animal almost always survives the infection. The prepatant

period is about 6 days and the animal soon becomes very ill

as schizogony is rapid, resulting in rapid multiplication

of the organisms. The animal becomes very weak 48 hours

before death, for it refuses food, and has pallor of the

skin and conjunctiva. There is high fever which becomes

subnormal shortly before death (Garnham, 1980).

^5

I n t r a v a s c u l a r h e m o l y s i s i s o f t e n p r e s e n t a long w i t h

h a e m o g l o b i n u r i a . t h e p h e n o m e n o n o f e x c e s s i v e b l o o d

d e s t r u c t i o n has been a t t r i b u t e d t o a u t o - i m m u n i z a t i o n

(Zuckerman. 1966) .

AIMS AND OBJECTIVES ̂

26

The present study aims to gheck the efficacy of P.

knowlesi antigens for their use in serodiagnostic testa for

human malaria. The role of serodiagnosis in malarial

infections is indeed limited. At best it could be used as

an adjunct to microscopic detection of parasites. Yet it

would make things simpler if P. knowles i antigen, which

can be easily obtained compared to antigens from human

malarial parasites, could be used for identification of

malaria specific human antibodies. Antigen pools from P.

knowlesi could then be used for conducting large scale

sero-epidemiological surveys in malaria endemic regions.

Present investigations were, therefore, aimed to test

the usefulness and the efficacy of P. knowlesi antigens in

the identification of human malaria antibodies by means of

indirect haemagglutination tests. If this antigen proves

useful in IHA test then, hopefully, the antigen from monkey

malaria can be usefully employed in several other

diagnostic tests such as ELISA, RIA and several others.

Slide smears with P. knowlesi infected monkey RBCs can also

be effectively used for the detection of malaria specific

human antibodies in IFA tests. Infact some preliminary

tests along these lines have already been done in one or

two other laboratories.

11

The Dresent investigations were therefore designed to

achieve the above objective of using P. knowTesi antigen in

IHA tests. The details of the experimental orotocols which

were followed are as under:

(1) P. knowlesi cell line was maintained in vivo using

Rhesus monkeys as an animal model.

(2) P. knowlesi antigen was isolated from the parasites

harvested from the blood of infected Rhesus monkeys.

(3) Serum samples were collected from clinically

diagnosed malaria patients irrespective of their

having a negative or a positive blood film.

(4) Serum samples were collected from patients with no

clinical evidence of malaria for the past five years

and negative malaria blood films to serve as

controls.

( 5 ) Indirect haemagg1utination tests were performed on

the serum samples using P. knowles i antigen for

serodiagnosis of malaria.

MATERIALS AND METHODS

28

ANIMALS

Two young and healthy four month old Rhesus monkeys

(Macaca mulatta) . a male and a female, each weighing about

3-5 kgs were purchased from a local animal dealer.

CELL-LINE

PI asmod i urn K n o w l e s i c e l l l i n e was o b t a i n e d f r o m t h e

M i c r o b i o l o g y D i v i s i o n o f t h e C e n t r a l D r u g R e s e a r c h

I n s t i t u t e . L u c k n o w , I n d i a .

ANTICOAGULANT

A c i d c i t r a t e d e . x t r o s e was o b t a i n e d f r o m t h e B l o o d

B a n k , J . N . M e d i c a l C o l l e g e , A l i g a r h .

STAIN

Leishman stain was purchased from BDH. England.

CHEMICALS

Sodium corbonate. sodium potassium tartarate.

barbital, sodium barbitone, copper sulphate, disodium

hydrogen phosphate, dihydrogen sodium phosphate and sodium

chloride were obtained from BDH (India). Tannic acid was

purchased from W.J. Bush and Co., England and Methanol from

Merck (India). Bovine serum albumin and saponin were

obtained from Sigma chemical Co., USA and chloroquine

29

injectible from IPCA Pvt. Ltd., Bombay Sheep red blood

cells were obtained from healthy sheeps in the Central

Animal House of the J.N. Medical College, Aligarh.

Sodium azide, used as a preservative, was obtained

from Rield, Germany.

GLASSWARE

A l l t h e g l asswa re used were o b t a i n e d f r o m Pyrex and

Co .and C o r n i n g ( I n d i a ) L t d . The m i c r o t i t r e " U ' b o t t o m

p l a t e s were f r om Cooke E n g i n e e r i n g C o . , A l e x a n d r i a .

G l a s s d i s t i l l e d w a t e r was u s e d t h r o u g h o u t t h e

e x p e r i m e n t and a s e p t i c c o n d i t i o n s m a i n t a i n e d w h e r e v e r

n e c e s s a r y .

SPECIMENS

Five ml of blood was taken from 50 patients with

clinically diagnosed malaria, who were attending the Out

Patients Department, or from those patients who were

admitted to the J.N. Medical College, Hospital Aligarh,

India.

The blood was drawn by a sterile syringe and needle

(21G) and allowed to clot in a sterile universal bottle, or

screw capped tube, which was later stored at 4*̂ C overnight.

The serum was obtained by centrifuging at 1,000 rpm for 30

30

minutes and stored in a sterile screw capped tuPe at -20°C

for further use.

Five ml of blood was also taken from 25 patients of

the same age group with no clinical evidence of malaria for

making malaria negative blood films to serve as controls.

MAINTENANCE OF Plasmodium knowlesi CELL LINE

The P. knowlesi cell line was maintained and allowed

to multiply In v i vo in Rhesus monkeys which were

inoculated, intravenously, with approximately 5.3 x 10

parasitized monkey erythrocytes. After the appearance of

parasites in the blood the parasitaemia reached B0% on the

5th day of their appearance; the animals were bled at this

stage and the blood collected in Acid Citrate Dextrose.

Five ml of the infected blood sample was inoculated into a

fresh animal, for subsequently maintaining the cell-line,

while the rest was pooled for antigen isolation. Normal,

uninfected monkeys were used as controls.

COUNTING OF PARASITES

Blood films were made every day by a finger prick to

observe the appearance of the parasite after which they

were used for counting the parasites.

31

STAINING AND FIXATION OF BLOOD FILMS

Fixation of the dried blood films was done by

using methanol for 4 minutes and the films dried again.

The staining was carried by mixing equal volumes of

Leishman stain and phosphate buffer (0.01 M, pH 7.2) and

then layering this solution on the fixed and dried blood

film for 30 minutes. The slides were then washed with

phosphate buffer, air dried and examined under the oil

immersion of a microscope (Carl Zeiss, Jena).

ANTIGEN ISOLATION

Parasites were harvested according to the procedure

by Spira and Zuckerman (1962). At 80S$ parasitaemia blood

was collected from the infected monkey in ACD by cardiac

puncture under anaesthesia (Pentabarbitone sodium). The

pooled cells were washed by centrifugation in chilled

normal saline. The buffy coat containing leukocytes,

plasma and platelets was discarded and the sediment with

normal and parasitized erythrocytes pooled. The pooled

erythrocytes were washed 4 times by centrifuging at 1500

rpm for 10 minutes and incubated with an equal volume of

saponin (0.05X in normal saline) with continuous stirring

in an ice bath for 10 minutes. The lysate was centrifuged

at 1500 rpm for 10 minutes at 4*^0, the supernatant

32

discarded and the pellet washed again 3-4 times with

chilled normal saline to remove erythrocytes. After the

final washing the pellet consisting of malaria parasite was

suspended in chilled normal saline.

PREPARATION OF WATER SOLUBLE WHOLE ANTIGEN

The parasites were disrupted by ultrasonication of

the parasite suspension in 3 stretches for 6 minutes

altogether, in a 50 watt, 9 Kc magnetostriction oscillator.

The suspension was centrifuged again at 1500 rpm for 20

minutes and the supernatant collected was stored as soluble

antigen,

PROTEIN ESTIMATION

Protein estimations were done according to Lowry ejt

al . . (1951) using a Bausch and Lomb Spectronic 21

Spectrophotometer at room temperature.

MEASUREMENT OF pH

The pH was measured by an Elico LI-120 pH meter using

sodium tetraborate (0.1 M, pH 8.18) and Potassium hydrogen

pthalate (0.05 M, pH 4.0) as standard buffers.

INDIRECT HAEMAGGLUTINATION TEST (IHA)

The IHA test was done according to the method

described by Mathews §:t al-, (1975).

33

Sheep erythrocytes (SRBC) were washed 4 times with

phosphate buffered saline (pH 7.2) after which a 0. 3S6 (v/v)

suspension of the SRBC was prepared using PBS. The

suspension was mixed with an equal volume of 1/20,000

tannic acid and incubated at 4*̂ 0 for 30 minutes with

intermittent shaking. Tanned SRBC were washed thrice with

PBS-BSA for the removal of free tannic acid and resuspended

at 3%. A 0.1 ml tanned SRBC suspension was mixed with an

equal volume of the diluted antigen and incubated at 37°C

for 30 minutes with intermittent shaking. The antigen

coated SRBC were washed 3 times with PBS-BSA and was used

for making a ^ .5% suspension.

An 0.025 ml of serially diluted serum sample was

added to an equal volume of antigen coated SRBC suspension

in a 'U' bottom microtitre plate. The control wells

containing tanned RBC, normal sera and antigen were

included in the test. The plates were incubated at room

temperature in a humid chamber for 1 hour and then at 4°C

overnight. The highest dilution of the test serum giving a

positive carpet like pattern was recorded as the end point.

Both positive and negative controls were used along

with the test samples.

34

TREATMENT OF INFECTED ANIMALS

5 mg/kg body weight of Chloroquine was injected to

all experimentally infected monkeys before the animals

reached the stage of maximum parasitaemia.

RESULTS

35

Blood films made from peripheral blood of P. knowlesi

infected Rhesus monkeys showed the presence of parasites on

the 7th day following intravenous inoculation of

parasitized monkey erythrocytes. The parasitaemia reached

to a maximum count of about 80% on the 12th day. In the

maximum diseased stage the animals became critically ill

showing hyperthermia; these animals became weak, pale and

refused all kinds of food. The animals were treated before

they had maximum parasitaemia and the parasites were not

seen in the peripheral blood after 2 days of treatment.

Out of the 50 clinically diagnosed malaria patients

21 (^2%) had a positive blood film and 29 (58%) had a

negative blood film. When the IHA test was performed on

the 50 clinically diagnosed patients, 17 (81%) out of 21

blood film positive patients gave a positive IHA test and

21 (73%) out of the 29 blood film negative patients gave a

positive IHA test (Tableil). The 25 normal control

patients, which were all blood film negative, showed a

positive IHA test in only 1 (4%) patient (Table:II).

The 50 malaria patients consisted of 23 males and 27

females: the total number of blood smear positive were 14

36

(67X) and 7 (33%) f o r ma les and f e m a l e s r e s p e c t i v e l y

f T a b l e : I I I ) .

Of t h e 21 s l i d e p o s i t i v e p a t i e n t s 18 (865K) we re

i n f e c t e d w i t h P. v i v a x and 3 (14%) were i n f e c t e d w i t h

P. f a l c i p a r u m ( T a b l e : I V ) .

37

Table I: Seropositivity of malaria antibodies in clinically

diagnosed malaria patients using indirect haemagglutination

test (IHA)

GROUPS TOTAL NUMBER TOTAL NUMBER PERCENTAGE POSITIVE BY IHA

Blood film positive

Blood film negative

21

29

17

21

81

73

Total 50 38 76

FIGURE 1: SEROPOSITIVITY OF MALARIA SPECIFIC ANTIBODIES BY IHA TESTS IN THE BLOOD FILM POSITIVE & NEGATIVE CASES

Total Number

38

(H

* *

Total number of blood film positive cases.

Total number of IHA oositlve cases in the blood film positive group.

Total number of blood film negative cases.

Total number of IHA positive cases in the blood film negative group.

39

Table II: Seropositivity of malaria antibodies in normal

control patients using IHA test

GROUP TOTAL NUMBER TOTAL NUMBER PERCENTAGE POSITIVE BY IHA

Normal 25 Control

40

Table III: Sex distribution of s l i d e p o s i t i v e m a l a r i a

patients

SEX TOTAL NUMBER TOTAL NUMBER OF PERCENTAGE SLIDE POSITIVE

Male 23 14 6 7

Female 27 7 33

TOTAL 50 21 42

FIGURE 2 : Photograph of Indirect Haemagglutination test

41

t » • • • • ^̂ r -2 . ^ %'%%%: .^

'̂9 • # # '̂ • -̂ '• ' ' • # # # # # - ^ •

r### « . . . . ^ • • • # # • • •

? • # # • •

^ # • • • • • • •

c • • * 4 • • • • ^ • • • • • • • «

- ^

^>

- >

, ^

, •

» 1

• )

42

43

Table IV: Distribution of Plasmodium vivax and Plasmodium

falciparum in slide positive malaria patients

TOTAL NUMBER NUMBER OF NUMBER OF POSITIVE P. vivax P. falciparum

21 18 3

PERCENTAGE 86 14

44

FIGURE 4: DISTRIBUTION OF P. VIVAX AND P. FALCIPARUM IN BLOOD FILM POSITIVE

MALARIA PATIENTS

p. vivax 86%

P. falciparum 14%

...J

45

Table V: Distribution of IHA titres in malaria patients

GROUPS TOTAL TOTAL INDIRECT HAEMAGGLUTINATION TITRES NUMBER NUMBER TESTED POSITIVE < 16 16-64 128-512 > 1024

(> 16)

Blood Smear 21 17 4 6 10 1 Positive

Blood Smear 29 21 8 13 8 0 Negative

Control 25 1 24 1 0 0

FIGURE 6: FREQUENCY OF IHA TITRES IN MALARIA CASES AND CONTROL

) 1024

C. IHA t i t res in blood film negative cases.

B. IHA t i t res in blood film positive cases.

A. THA t i t res in Controls.

DISCUSSION AND CONCLUSION

47

Until some time back the only source of malaria

parasites was the infected host; however, the development

of continuous j_n vitro culture of P. falciparum has lead to

a large-scale production of infected blood stages in vitro.

Malaria parasites have various degrees of host

specificity and pathogenicity which affect the level of

parasitaemia obtained in any species. The degree of

infection with a given parasite may be affected by factors

such as age, sex and genetic strain of the host (Zuckerman

and Yoeli, 1954; Eling et al. , 1977; Miller 1976). Blood

collected from infected animals or humans which is to be

used for harvesting Plasmodia, may be subjected to certain

preliminary treatments to remove unwanted blood components

and to increase the proportion of parasitized erythrocytes.

Plasma is removed by centrifugation and washing, the buffy

coat is removed from the packed red cell mass to reduce the

number of platelets and leukocytes (Zuckerman et, al . .

1967). Leukocytes may be removed by suspending the washed

blood cells in several volumes of dextran solution or

obtaining them as supernatant on sedimentation (Zuckerman

et aj.. , 1967, Langer et aj.. , 1967). Sedimentation velocity

of washed blood cells on dextran (Levy and Chow, 1973) or

on sucrose density gradient (Williamson and Cover, 1966) or

on Ficol1-Hypaque solution which usually removes about 75^

leukocytes (Wallach and Conley, 1977). The slow speed

centrifugation, which we used in our experiment, removes

about 85* leukocytes. Homewood and Neame M976) reported

that the most efficient procedure for removing leukocytes

is the passage of diluted blood through a column of filter-

paper powder.

P. knowlesi infection in the Rhesus monkey (Macaca

mulatta) follows a characteristically rapid and fatal

course resulting in the death of host by day 10 post-

inoculation (Dutta et al., 1981). While running a typical

course of infection, it was revealed in differential

parasite counts that the infection in Rhesus monkey is

rather synchronous at a particular point and at this time

the parasites mostly appear in the same stage of

development (Khanna, 1984).

Resurgence of malaria has currently made us realise

the importance of developing some reliable serological

tests. Serology is an important epidemiological tool along

with blood slide examination, as malaria cannot always be

detected by direct microscopic examination of blood film.

Detection of antibody to malarial parasites can be useful

in the diagnosis of clinical cases as well as screening for

49

disease c a r r i e r s , mainly in n o n - e n d e m i c a r e a s , and in

epidemiological studies (Agarwal et aj.. , 1982; Biswas et.

a1 •, 1990). The microscopic examination of blood slides is

not the only parasitologic l a b o r a t o r y p r o c e d u r e used in

malaria surveillance work to determine the occurrence of

the disease. This method can only indicate the presence or

absence of patent parasitaemia at the time of examination;

it does not indicate the individual's malaria experience or

the state of his immunity CKagan, 1972).

Serologic tests are needed to detect infections in

those cases in which the parasites in the peripheral blood

are in such low numbers that they cannot be detected

readily by parasitological examinations. We need serology

to determine whether eradication measures have been

successful, that is, whether transmission of malaria in

endemic areas has been interrupted. Moreover, a proper

surveillance needs to be maintained in those regions in

which malaria has been successfully eradicated in order to

prevent reintroduction of the parasite, which could bring

about devastating epidemics in populations rendered

susceptible by the absence of infection (Sadun, 1972). A

practical serologic method for use in malaria programs must

meet the following criteria, (a) the test has to be simple

to perform; (D) the interpretation of results must be free

50

of subjectivity; (c) the test must be rapid; (d) the cost

must be minimal; (e) the tests results have to be

sufficiently sensitive and specific, and (f) the test must

be capable of producing repeatedly reliable results in

various laboratories. The indirect haemagglutination test

meets these criteria better than other available serologic

methods like indirect fluorescent antibody test and the

complement fixation test (Kagan, 1972).

The IHA test may have a wide range of applications in

epidemiology. For example it may be used (1) to measure the

level of malaria endemicity (Kagan et al. , 1969a); (2) to

verify the absence or presence of transmission from a

relatively small number of specimens (Mathews et. al . ,

1970a); (3) to delineate malarious areas (Mathews et al . .

1970b); (4) to detect seasonal changes of malaria

transmission (Kagan et aj.. , 1969b); (5) to investigate

suspected reintroductions of malaria into consolidation or

maintenance phase areas; and (6) to detect small foci of

malaria transmission (Mathews ei al., 1970b). The test is

especially useful in areas where the regular malaria

surveillance work is limited, and it can be used

effectively as a screening mechanism to detect potential

carriers of malaria.

51

P. falciparum antigen obtained from infected Actus

monkeys (Voller et al-, 1976; Mahajan, et al-, 1981), or

from i_n vi tro cultures (Spencer e_t_ aj_.,1981) has been

successfully used for diagnosis of malaria by the IHA test

(Farshy and Kagan, 1972; Wilson ejt aj.. , 1975). Due to

limitations in the availability of adequate amount of

P. faici parum antigen in a sufficiently purified form,

efforts are being made in several laboratories to evaluate

the possibility of using P. knowlesi antigen for diagnosis

of human malaria (Chandanani et aJ.-. 1981; Agarwal et al . .

1981). This Plasmodium species was selected because Rhesus

monkeys can be readily infected and can provide a large

volume of parasitized red blood cells for obtaining antigen

to be used for detection of P_. f al ci parum or P.. v i v a x

infections (Kagan, 1972; Agarwal et al-, 1983).

Our results show that 81% of the slide positive

malaria patients obtained a significant antibody titre of

2 1:16. This finding compares well with the of Agarwal et,

al (1981, 1982) and Marwah et. aJ.. (1989); who also showed

75%, 84.5% and 78% of significant antibody titres in slide

positive malaria patients respectively. The slide negative

patients showed a significant antibody titre in 73%, this

seems to be in contrast to Marwah et, aj_ (1989) and Agarwal

et al (1982) who showed 32% and 7.14% positive antibody

52

titres. The reason for this difference could be because

most of the patients attending J.N. Medical College

Hospital come from places in and around Aligarh which are

at some distance from Aligarh and as such they do not come

back again for slide parasitology; drugs are given to them

on the basis of clinical symptoms of malaria irrespective

of a negative laboratory diagnosis. The slides are

generally made at a time when the parasites in the

peripheral blood are in low numbers and as such the

parasites are not easily detected. Differences in the

method for preparations of P_. knowl es i antigen and the

storage conditions may explain the low titres reported by

Agarwal et al (1982) and Marwah et al (1989).

The sera specimens from the 25 healthy control group

were also subjected to IHA tests. Only 1 (4^) sample gave

an antibody titre of >_ 1:16, this is in accordance with the

results of Agarwal et aj. (1981; 1982) who showed positive

IHA values in 4% and ^ . 5% respectively. A positive IHA

result in 15.5* of normal healthy subjects has also been

reported by Srivastava et aJ. (1983).

In this study the sera were collected from 23 males

and 27 females and out of these 61% males and 335̂ females

were found to have a positive blood film. Out of the 50

slides made from the blood of clinically diagnosed malaria

53

patients only 21 slides showed malaria parasites giving a

slide positivity rate of 42%: this is a little lower than

that reoorted by Chowdhury ejt a_i (1983) who reported a

slide positivity rate of 67.5%. This could be because the

samples were collected from February to September and the

highest incidence of malaria being from September to

November only, and seemingly as such, the tip of the

iceberg was reached (Chowdhury et al., 1983).

Of the 21 slide positives 18 were of P. vivax and 3

of P. f alci parum; this can be compared to the work of

Marwah et al (1989) who also showed a higher incidence of

P. vivax (94X). But these findings are in contrast with a

study done in Senegal where P. falciparum was found to be

the predominant species (Vercruysse et aj.. , 1983).

Our results show that the IHA antibody titres of

slide positive malaria patients ranged from l 1:16

to 2 1:1024 with the highest number of positives having

titres between 1:128 to 1:512. In the slide negative

malaria patients most of the subjects having a positive IHA

antibody titre ranged from 1:16 to 1:64 titre. The 4 of

the slide positive sera gave a titre <_ 16, such a low titre

could be attributed to the fact that IHA test is rather

insensitive at detecting antibodies in the early phases of

54

primary infection. No slide negative patient had a

titre >. 1024 (Marwah et al-. 1989).

On the basis of this investigation and in the light

of other similar studies it can be concluded that antigen

prepared from £. knowlesi parasites can be successfully

employed in the IHA test for detecting malaria specific

human antibodies. An IHA positivity of 81% has been seen

in the present study and this is in agreement with the

results reported by other workers using heterologous

ant i gen (Lobel e_t al . , 1973; Agarwal ejtaj.., 1981).

Apparently, a high degree of specificity resides in the

test as was evident from the very low positivity (4%)

obtained among normal healthy controls. An IHA positivity

of 73% in patients with slide negative malaria suggest that

a small number of such persons may indeed be suffering from

malaria. If serology is to be employed, as an adjunct to

microscopic detection of parasites, the, the diagnosis of

clinical cases of malaria on the basis of IHA antibody

titres can be conveniently carried out on more or less a

routine basis.

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