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Immunofluorescence and the measurement of immune response to hyperendemic malaria
395 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL _VIEDICINE AND HYGIENE.
Vol. 59.
No. 4. July, 1965.
COMMUNICATIONS IMMUNOFLUORESCENCE A N D T H E M E A S U R E M E N T OF R E S P O N S E TO H Y P E R E N D E M I C MALARIA
IMMUNE
~Y I. A. McGREGOR AI~I)K. WILLIAMS The Medical Research Council Laboratories, Gambia, West Africa
A. VOLLER London School of Hygiene and Tropical Medicine AND
W. Z. BILLEWICZ* Medical Research Council, Obstetric Medicine Research Unit, Aberdeen Rates of splenomegaly and parasitaemia today form the basis for the measurement of malarial endemicity. Neither method is entirely satisfactory; enlargement of the spleen may be caused by other common diseases, and the incidence of parasitaemia alone can utterly faR' to present an adequate picture of the pattern of malaria in a population. For more than half a century it has been appreciated that the manifestations of malaria are largely governed by the degree of immunity possessed both by the individual and by the community (Ross, 1910). Any method, therefore, capable of measuring the immune state with even moderate accuracy would be of great importance in advancing our knowledge of malarial epidemiology, and would have an immediate application in the assessment of progress of eradication campaigns. Field studies made largely in Africa over the past 15 years have revealed a constant relationship between the rising concentration of serum gamma globulin and the acquisition of clinical immunity to plasmodial infection (MCGREGOR et al., 1956; MCGREGOR and GILLES, 1958 and 1960; GILLES and McGREGOR, 1959 and 1961; EDOZlEN, 1961) while, more recently, the passive transfer of malarial immunity in man has been demonstrated (COHEN, MCGREGOR and CARRINGTON,1961; COHEN and MCGREGOR, 1963). Over approximately the same period the serological specificity and potentialities of immunofluorescence have elsewhere been studied (CooNs, CREECH and JONES, 1941; WELLER and COONS, 1954). Lately these two fields of interest have converged and the measurement of specific antibody in the blood of malarious subjects by fluorescent immunological methods has been reported (KuVlN et al., 1962; VOLLERand BRAY, 1962). In this communication are reported the results of an attempt to measure by an immunofluorescent technique the levels of malarial antibody possessed by African subjects of different ages, resident in a hyperendemic environment. The investigations were made in January 1964 in 4 Mohammadan, Mandinka villages--Keneba, Jali, Manduar and Kantonkunda--situated in the West Kiang district of the Gambiaman area which has already been fully described (McGREGOR and SMITH, 1952; BERTRAM,McGREGOR and McFADZEAN, 1958). The study area is small and may * The authors gratefully acknowledge the financial support provided by the World Health Organization for this study. C
396
IMMUNOFLUORESCENCE AND IMMUNE RESPONSE TO HYPERENDEMIC MALARIA
be confined within a circle 5 miles in radius, centred upon Keneba. Within it the principal vectors are the fresh-water-breeding A. gambiae gambiae and its salt-water variant A. gambiae melas. Malaria, although hyperendemic, is not uniformly transmitted throughout the year. Mosquito density rises sharply before the onset of the rains in June, and initial populations are almost exclusively A. gambiae melas. As the rains progress, A. gambiae gambiae gradually replaces its variant and by August constitutes the dominant anopheline mosquito. Later, when the rains are about to cease, usually September-October, A. gambiae melas resumes dominance for a short time. While populations of A. gambiae gambiae seem evenly distributed throughout the 4 villages studied, those of A. gambiae melas appear to vary inversely with the distance of each village from the saline swamps. By January of each year the advent of hot and exceptionally dry weather drastically reduces anopheline density to low levels, which are then maintained until the following May. The transmission of malaria rises sharply in July and August and is maintained at a high level until November or December. Thereafter it declines and remains low for the rest of the year. Thus, when the investigations to be described were made, the human population had just emerged from the period of peak transmission and was entering the relatively non-malarious season.
Subjects and methods By a system of birth recording maintained for research purposes by the staff of the laboratories, the ages of all children under 5 years in the 4 villages were known with precision. Further, for the previous 2 years each available child in the study area had been examined at intervals of 3 months, when its general health, haematological indices, and malarial state had been assessed. Governed mainly by the unique facilities at hand, the primary objective was to determine the fluorescent antibody titre of each child under 5 years of age resident in the 4 villages, and to consider the results obtained against the available malariometric data. Accordingly 320 children were examined; the height and weight of each were recorded, health and nutritional states assessed, haemoglobin, packed cell volume and mean corpuscular haemoglobin concentration values determined, thick and thin blood films made for parasite identification and enumeration and, from fingerprick blood, a plasma sample was obtained for subsequent fluorescent antibody assay. A secondary objective was to determine fluorescent antibody levels in samples of older age groups, and to this end 16 children of 5-10 years, 26 of 11-16 years, and 84 adults were examined. The children aged 5-16 years were from Keneba only, since birth recording had been in operation longer there than in the other villages and since it was important to know the ages of subjects with accuracy. Of the adults, 18 were resident in Manduar, 17 in Jali, and 49 in Keneba; 44 were males and 40 females; of the latter 12 were pregnant. The older subjects were not medically examined; samples of blood for fluorescent antibody assay were taken by venepuncture and thick and thin blood films prepared from each subject. Thick blood films were stained by Field's method, and thin films after alcohol fixation, by Giemsa's. Parasites were enumerated against leucocytes and densities calculated by assuming a standard leucocyte count value of 8,000 per c.mm. 100 oil fields of each thick blood film were examined. Finger-prick blood samples were collected in heparinized capillary tubes, a minimum of 2 being taken from each subject. After sealing, the tubes were spun in a microcentrffuge for 5 minutes, the packed cell portion was broken off and the plasma made up in phosphate buffered saline to a dilution of 1:25 and stored at --20°C. until tested. As a check to the similarity of results obtained by finger-prick and venous blood collection, samples by both methods were taken from 20 adults. Comparison of antibody assay in these samples was satisfactory; in no
I. A. MCGREGOR~ K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
397
instance did results vary by more than one dilution--a variability not infrequently encountered in determining the end point of fuorescence in the serial dilution of any single serum sample. Thin blood films from patients exhibiting P. falciparum parasitaemia of not less than 30,000 trophozoites per c.mm. constituted the antigen. Films were air-dried over calcium chloride for 24 hours and then stored at --70°C. until required. They were then dehaemoglobinized in N/3 hydrochloric acid and, after washing with phosphate buffered physiological saline (pH 7" 2), layered with the appropriate dilution of plasma or serum under examination. After permitting interaction for 40 minutes, the film was again washed in buffered saline, dried and layered with fluorescein-labelled antihuman-globulin serum. 30 minutes later it was again washed in buffered saline. Finally, it was examined by fluorescence microscopy and the effective titre was taken to be that dilution at which fluorescence was last detected. The technique employed permitted titre assessment within broad groups only. Thus a titre reported as less than 1:25 embraces those from zero to 1:24, while those classified as 1:25 include dilutions in the range 1:25-1:49. For purposes of numerical analysis logarithmic transformation of titre has been used to normalize distributions and stabilize variances. Results
A. Results considered irrespective of village of residence Titre distribution within age groups is shown in Table I, while mean logarithmic titres for each age group are expressed graphically in Figure 1. These data clearly show that, after the early months of life, the antibody content of blood increases as age advances. In Table I, while a few relatively high values are recorded, the
MEAN
LOG TITRES (446
BY AGE GROUPS
subjects)
3.5"
30' . I
2.5' J
2.0
/
,../ 1.5, i
I
,s
|
¢"
(Mean ocje=33yeors)
Age
in y e a r s
Fro. 1.
398
IMMUNOFLUORESCENCE
AND
IMMUNE
RESPONSE
TO
HYPERENDEMIC
MALARIA
titres o f children aged less than 5 years are scattered fairly uniformly at the lower levels of the scale. For adults, values tend to be higher and there is a total absence o f low values. The relatively rapid decline in antibody levels in the months immediately after birth, probably indicating the decay of passively acquired immunity, is evident in Figure 1. Levels remain low until the end of the first year of life and thereafter increase throughout childhood into adult life. T h e rate of increase appears to be relatively fast in early childhood, and to become slower in older childhood and in adult life. TABLE I.
F.A. titres of the entire young child population (0-4 years) and of samples of older invidividuals resident in Keneba, Jali, Manduar or Kantonkunda.
Number Age ~ in Group Group <25 25 Months 0-
F.A. titre 50
Log titre
100 200 400 800 1600 3200 6400 Mean
S.D.
] 5
1
1]
2
2.6577 0"3432
1
I
1-
5
2
1
2-
11
4
2
3
--1"--~0620'3914
3-5
17
4
4
2
7
1.7451 0.4424
6-8
17
4
5
2
5
1
9-11
12
3
2--7
2
2
i] I
34
3
i 8-T
12
8
"
18-23
49
5
2.0660 0.4872
24-35
5
i 81 4- - - - V17m m11
3 .
m i62n i
2-1862 0.4154
36-47
57
2
48-59
51
Years 5-10
16
12-17
,
2
' 1.9955
0.4565
1.7274 0.4555
26
17
6 i
1-7809 0.5043 1
2-0189 0.4695
6
4
18
20
5
2
2.2438 0.4196
4
4
15
21
5
2
2.3237 0-3478
6 [ 4 I. 10 ! 8
2- 7405 0.2427
i
6
i
11-16
26
1
3
17 +
84
1
11
20
31 108 103
57
Total
446
28
44
4
2.9055 0.3090
26
20
3.0326 0.3544
45
24
In Figure 2 the individual titres of 25 infants aged less than 16 weeks are shown. Again, the rapid decline from high values in the first and second weeks o f life is apparent. Such a pattern supports the view that, in man, antibody transfer from mother to child is solely prenatal and transplacental (BRAMB~LL, 1958; BA~GHAM, 1960). It also suggests that the rate of decay of passively acquired immunity may be faster than is generally thought, and it should be recalled that, as some o f these infants had recently emerged from a season o f frequent malarial transmission, the rapid fall may have, in part, been the result of antibody utilization.
I. A, MCGREGOR~ K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
INDIVIDUAL TITRES OF 25 INFANTS
AGED LESS THAN
399
16 WEEKS
800700 600 500400300200IOO-
+- + ++++++ I L 2
3
5
4
;
-,
8
+
T
i
Io
,t
T
T
T
12
Age in weeks FIG. 2.
TITRE DISTRIBUTION WITHIN AGE GROUPS (.320 CHILDREN 5 YEARS OF AGE) 50
_ 0 - 3 months ( 2 5 )
30 t
2 4 - 3 5 months (62)
32.oo
30
t+oJ +oo
I0
'°1 I t I ~
4-11 months C42)
~.o+
~5o1 o -i
33-33
?.+,e l
I
I '.+' 3 6 - 4 7 months
(57)
23.8, 2r.,,,
30
0
2"p..t
u.~
- o WlO ", 0
.
50
1 2 - 2 3 months ( 8 3 )
I0 53
1~-77
3 5j
[3.s~ I I
48-59
C
months ( 5 1 ) 4118
u~ 3501 0
19 2 8
I0
9.64
22.89 7.23
3 6!
:~.41 '
~ ¢ ~ ° ° ~~
.? o ° o
0
I0 I
0 Titre FIG. 3.
~.q'~
0
0
0
400
IMMUNOFLUORESCENCE
AND IMMUNE
RESPONSE TO HYPERENDEMIC
MALARIA
In Figure 3 the distribution of titres of children under 5 years of age is shown. To counteract the effect of passively acquired antibody the infants are divided into two groups, 0-3 months and 4-11 months; thereafter older children are grouped by year of age. Again the general trend, manifest in a gradual shift into higher levels as age advances, suggests a slow consolidation of titre. The slowness of the change is obvious from the broad similarity of the histograms relating to the 3rd, 4th and 5th years of life, as well as from Figure 1. The specificity of the fluorescent antibody technique as applied to malaria has been demonstrated by KuvIN et al. (1962). It is necessary therefore that the titres under description should be considered in relation to the malarial experience of the subjects studied. In 28 of the 320 children under the age of 5 years antibody could not be demonstrated at a plasma dilution of 1:25. Table II comprises a list of these children and gives for each the age and malarial experience as judged by quarterly blood film examinations, wherever these were available, in the 2 preceding years. For 18 of the group no evidence of any malarial infection could be established; in the remaining 10, infection was definitely known to have occurred. Of these latter, only 3 harboured trophozoites in the circulating blood at examination in January 1964, and only 2 others were trophozoite-positive in November 1963. The table therefore indicates a strong correlation between low, even negligible, antibody concentrations, and the absence of recent malarial infection. In subject M.42 the year of apparent freedom from asexual parasitaemia which followed a year of ostensibly continuous infection may have been associated with the progressive decay of any immunity acquired. For patients J.52 and J.53 no explanation of the low titres observed can be offered. T~Lo. II.
The malarial experience, as judged by quarterly blood film examination, of children possessing F.A. titres < 1/25 Parasitaemia
Village Keneba K. 107
Age 1962 years months Jan. Apr.
1963 1964 July Nov. Jan. Apr. July Nov. Jan.
(Jan. 1964)
2
Neg. Neg.
65
5
Neg. Neg.
47
6
Neg. Neg.
43
8
Neg. Neg. Neg.
98
I
1
Neg. Neg. Neg.
2
71
5
57
6
36
11
64
N.S.N.S.N.S.
N.S. ! N . S . N . S . N . S . N . S .
Neg. Neg. 3
7 L
Neg.
Neg. Neg. Neg. Neg. Neg. Neg. I
io 4O
÷
+
÷ Gam.
+
+
Neg. Neg.
+
÷ Neg. Gam.
+
Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. i
401
I . A. M C G R E G O R ~ K. W I L L I A M S ~ A. V O L L E R A N D W . Z . B I L L E W I C Z
TABLE II~continued. Parasitaemia ~
ViHage
Age 1962 years months Jan. Apr.
r
1963 July Nov. Jan. ii Apr.
1964 July Nov.
Jan.
!
Manduar M. 37
Neg. Neg.
1
Neg. N e g .
26
Neg.
9
1
Neg.
Neg. Neg.
Neg. 4 Neg. Neg. Neg. I Gam.
24
1
1
Neg. Neg. Neg.
29
1
1
Neg
17
2
42
2
1
N.S.
44
2
1
Neg. Neg. Neg. Neg. Neg. N e g . Neg. Neg. Neg.
20
2
3
Neg. Neg. Neg.
49
2
5
N.S. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. i Neg. Neg. Neg. Neg. Neg. !
Gam. +
+
Gam. Gam. ] ÷ + ]Neg. Neg. Neg.
+
J
+
+
Neg. i Neg. Neg. Neg.
Jali J.
55
Neg. Neg.
13
Neg. Neg.
98
Neg. Neg.
29 52 53
r Neg. Neg. Neg.
8 r I
1
11
3
3
IKan tonkunda K.K. 28
Neg. N . S . N . S . N . S . N . S . N.S. N.S. N.S.
+
+ t
+
Neg.
+
+
+
+
+
Neg. Neg.
N.S. - - not examined. For the children of Keneba comprehensive medical records exist, spanning the 2 preceding years. In this period all medicines administered to sick children were carefully noted. These records show that treatment for acute P. falciparum malaria was afforded to subjects K.40 and K.71. It is of further interest to note that the father of subject K.64 was an employee of the Health Services of the Gambia Government and that he had long been suspected of intermittently administering antimalarials to his child. This point, however, could not be proved. For the children of the other villages no
402
IMMUNOFLUORESCENCE AND IMMUNE RESPONSE TO I-IYPERENDEMIC MALARIA
comprehensive medical records exist but it is known that access to antimalarials could only have been sporadic, if indeed it occurred at all. The titre distribution in all children under the age of 5 years according to the presence (241 subjects) or absence (79 subjects) of parasitaemia at the time of examination, is depicted in Figure 4. Again the correlation between patent infection and increased titre is apparent. TITRE DISTRIBUTION IN CHILDREN
AGED < 5 YEARS
(ACCORDING TO P A R A S I T A E M I A )
40-
Blood film posttlV¢
30-
=
20-
~ _"
~ ~
co
~ 6,
c
-6.
; Or, 0
I0-
o
~c
,40"
~_
30"
Blood film negative
~ "/
at:) / 1 1
777-~
,/n~/Al--I~
~ l l Il l l Il
I
'25
25
50
I00
200
400
, 800
Titre
FIG. 4.
Figure 5 shows the titre distribution in gametocyte-positive (78) and gametocytenegative (163) infected children aged less than 5 years. It supplies little evidence to suggest that, in this age group at least, the presence or absence ofgametocytaemia is of any value as an index of immune state. An analysis of titre distribution according to density of parasitaemia for all children from 4 to 59 months of age is presented in Table Ill. It will be noted that titre distribution in the more heavily infected subjects tends to be restricted to the intermediate range, both high and very low titres being absent. However, this table gives no indication of relative rates of antibody synthesis and utilization, and it may well be that turnover rates in heavily infected individuals greatly exceeded those of their more lightly infected fellows.
I. A. MCGREGOR:, K. WILLIAMS, A. VOLLER AND W. Z. BILLEWICZ
TITRE DISTRIBUTION
IN M A L A R I O U S
403
CHILDREN
AGED 45 YEARS ~ACCORDING TO G A M E T O C Y T A E M I A ) T r o p h o z o i t e 4.v¢.
40-
Gometocyte -re. 30-
8
20-
co
~
0el
=0-
I0-
o
~ o
~dE
--I
o 40-
U L
o.
30-
20-
m
o,,
"
n./
- IAI/~IA
•
":
IO"
O
!
<25
!
25
,
50
1
IOO
i
200
i
400
I
800
Titr¢ F I G . 5.
In Table IV are shown the titre distributions according to the number of positive quarterly blood films recorded in 6 consecutive examinations for 153 children aged 24-59 months. Children under 24 months of age have been excluded since most were not of sufficient age to have been examined on 6 successive quarterly examinations and since these younger children tend to exhibit a titre pattern which is to a considerable extent age-confounded. It will be noted that the frequency of recorded parasitaemia does not appear to influence titre distribution, except in the 4 children possessing no record of infection in the preceding 18 months' period, all of whom show titres of less than 1:25. The results expressed in this table therefore strongly suggest that simple duration of parasitaemia is not the only, or possibly even the most important, factor in the progressive acquisition of immunity, and they should be considered in conjunction with those given in Table I I I . Together these suggest that neither density nor duration of parasitaemia is the single dominant factor in creating high concentration of antibody, and that consequently it is probably their combined effect that is important.
404
IMMUNOFLUORESCENCE AND I M M U N E RESPONSE TO HYPERENDEMIC MALARIA
TABLE III.
Titre distribution in children 4 to 59 months of age according to parasite density. F.A. Titre
No.
in 100 200 400 800 1600 group
<25
25
50
Blood film negative
21
16
4
8
4
2
Blood film positive; Parasites per c.mm. < 100
1
4
1
11
6
1
2
2
2
23
17
6
10
27
2
5
< 15,000
1
<20,000 > 20,000
< 1,000 <5,000 < i0,000
TABLE IV.
1
%
1 or2
%
3or4
%
5 6
S.D.
55
1.6108 0"5116
1
25
2"1570 0.4290
5
2
53
2.2887 0-3810
29
9
3
84
2.2979 0.3501
19
6
3
36
2.17120.3366
2
7
7
1
1
2
6
6
15
2"1833 0.3290
4
1
1
3r
9
F.A. titre <25
25
5O
100
Log titre
200
400
800
100.0 1
1
1
2
2
5 '9 3
2"9
11"8 4
8"8
6
9.
!7 11 "8
8
11 "8 6.3
1.5
Mean
S.D.
1-0969
--
100" 0
5"9
1
Total 4
4
o/ /o
%
Mean
Titre distribution according to number of positive quarterly blood films in six consecutive quarters (153 children aged 24-59 months).
No. of positive films None
Log Titre
3 41 "2
13
23"5
17 "6 1
4
38"2
5"9
11 "8
34 2.9
12 13 5 37.5 40"6 15 "6
17 3 1 7 10' 6 3045 •4 25 "8
2.2683 0.4181 99.9
32
2.3736 0"2491
100.0
2 4"5
17 2"3251 0.4917 100.1
66 3"0
2.2054 0- 3547 99.9
T h e range of parasite density within age groups is shown in Figure 6 for the 446 individuals studied, and the titre distribution for the same subjects is shown in similar fashion in Figure 7. T h e ability, as age advances, of the individual resident in hyperendemic circumstances to restrict parasite multiplication is clearly demonstrated i n Figure 6, and the concomitant progressive increase in antibody concentration is discernible in Figure 7. W h e n these figures are considered it should be remembered that, in the 0-4-years age
I.
A. MCGREGOR~
K. WILLIAMS~
dnoa6 Io 0
0
0
0
0
2,~.~.
~,~,~,
A. VOLLER
AND
W.
~Bo%u~d
0
00
0
=,~,~
0
0 0 "7
0 ,
,
0 ,
,
C-
o
°
~-
,n
~
7.
,--."
0
r--
~'
000
~ c
L~
r.-, ,
. . . . . .
u~
~
o~L°°~
-
~I
°F--
~ "~ I
~ ~ ~, ~ o
c~
IE
405
Z. BILLEWICZ
00~"
,
o
7
~,
-I
0
!
m
o
.
~'
.001
>
,,," m
"~ .-2
.-2
Ooe
0.9
x, .-
<
6'6'o
b'6'o dno~6
6'6'6
6'6'6 ~o ~ 6 o ' ~ u ~
d
01
t--i
~'
~_~
1
(.91
>-'
-00
zl
r.
"0 o
q co
v
r,.
o >. nl
m
m
0
I
I ol I--I
u
6 " ~ 6
d ' 6 6
~'~~
dnoJ6 ;o ~6o~u~J~d
%
o% %.;
Ioo.,
°oO>j
!
°o/,
~.~-~,o
406
IMMUNOFLUORESCENCE AND IMMUN'E RESPONSE TO HYPERENDEMIC MALARIA
group, are included members in the first few months of life who may not have experienced malarialinfection and who frequently possess high antibody levels inherited transplacentally. Figure 7 amply indicates how simple parasite rates fail to reflect the progress of the development of immunity. The similarity of such rates in the 3 young age groups obscures the considerable progress that is evident when parasite densities and antibody titres are considered. Viewed together, the findings illustrated in Figures 6 and 7 suggest that the antibody measured by the technique involved is intimately connected with antiparasitic immunity and may indeed be the specific antiplasmodial antibody. For many years the spleen rate has been regarded as an important malariometric datum, its magnitude in the age group 2-9 years being an index of malarial endemicity. In Figure 8 the titre range is contrasted of children 4-59 months of age with (226), and without (58), splenomegaly, while Table V relates titre to splenic size (Hackett's classification). Children in whom the accuracy of splenic palpation was dubious have been excluded. It will be noted that the frequency of low titres is considerably higher in children without splenic enlargement, and that increase in splenic size appears to be consistently associated with increase in antibody titre. The antibody titres in adults, classified by sex and parasite incidence and density, are shown in Table VI. TABLEV. F.A. titres of children 4 to 59 months of age according to splenic size (Hackett's classification) (Children whose spleens may have been imprecisely palpated are omitted). F.A. titre
Log titre
Group as No. percentage in of total group sample
--,[--
Size of spleen <25 25 0
14
12
1
4
6
2
5
13
1001200 400 800 Mean
50
3'191 5
1 1.8429 0"5363
58
20.42
2
2.0687 0"4526
43
15.14
46 i 10
4 2-2077 0'3896
153
53-87
2
1 2.3326 0.3596
25
8-80
2
2.4169 0-3298
5
1 "76
284
99" 99
i
57 .
.
.
3
7
4
3
TABLE VI.
1
8
1 4 , 12
18 .
S.D.
I
121
F.A. titres of adults according to parasitaemia at time of examination. F.A. titre
Group <25 25 5 0 Males Blood film neg.
Log titre in 100 200 400 800 1600 3200 group Mean S.D. No.
I
1
518
10
9
3
4
i
Blood film pos.
1
Females Blood film neg.
4
8 11
Blood film pos.
1
4
I 3
36
3"0290 0.3841 3" 1544 0"3116
1
26
2" 9403 0" 2978
2
14
3" 1222 0-3707
I. A. MCGREGOR~ K. WILLIAMS) A. VOLLER AND W. Z. BILLEWICZ
407
Though the group involved is small, the suggestion is apparent that women are represented in the higher titres to a lesser degree than men and, further, that patent parasitaemia is more frequent in them. Of the 40 female adults studied, 12 were pregnant, and of these 41.67% showed patent parasitaemia compared with 31.14% of the non-pregnant women. The titre distribution in these women is shown in Table VII.
TABLE VII.
F.A. titres of pregnant and non-pregnant women according to parasitaemia at time of examination. F.A. utre
Group <25 25 Pregnant Blood film neg.
50
Log titre in 100 200 400 800 1600 3200 group Mean S.D.
i
1
Blood film pos.
No.
3
2
3
2
1
7
2"9502 0"3830 2"8986 0' 1649
Non-pregnant Blood film neg.
3
4
10
2
Blood film pos.
1
1
1
4
2
19
2.9524 0.2714
9
3" 2464 0" 4014
Though mean log titres of pregnant and non-pregnant, unparasitized women are almost identical (2.9502, 2.9524 respectively), the mean log titre of pregnant, parasitized women (2. 8986) is substantially lower than that of parasitized non-pregnant women (3- 2464). With these small numbers, however, this difference, even although statistically significant, cannot be regarded as firmly established. The relationship of antibody titre to haematological indices (Hb, PCV, MCHC) was examined for children under 5 years of age. No pattern of improving haematological status with advancing antibody titre was observed. This result was not unexpected since in any child high antibody titre may well be the result of a recent severe attack of malaria. Thus anaemia and relatively high antibody titre must be expected to be simultaneously present in some proportion of young children. Among the 320 young children studied were 14 who were classified as being in poor nutritional state. The basis for such an assumption was a body weight well below that expected for the age of the individual, coupled with evidence of wasting and poor tone of the muscles. The relevant details are shown in Table V I I I and it will be noted that the antibody titres of these children provide no evidence to suspect that their nutritional state had materially affected their capacity to synthesize antibody. This finding accords with that of COHEN and HANSEN (1962), who found no evidence to indicate that the state of nutrition affected either the distribution or the turnover of gamma globulin in a group of children in South Africa. O f the 241 children aged less than 5 years known to be parasitized, 219 were infected with a single, 16 with two and 6 with three, species of plasmodia. Contrast of titres in these subjects revealed nothing which could be interpreted as indicating that multiple infections were associated with higher antibody levels than was infection with a single species.
408
IMMUNOFLUORESCENCEAND IMMUNE RESPONSE TO HYPERENDEMIC MALARIA TABLE V I I I . Village
Antibody titre in children with signs suggestive of malnutrition. Sex Age Years Months
Weight lb. oz.
Hb G.
Titre
Condition
i
M
15
15
9'0
100
Marasmic
Kantonkunda
M
28
1
9.7
100
Muscle tone poor, some wasting
Kantonkunda
M
14
12
12.4
100
Pronounced muscle wasting
Manduar
M
16
13
7.8
400
Pronounced muscle wasting
F
11
13
7.0
100
Marasmic
F
12
11½
11.7
25
Muscle wasting, tone poor
M
16
15
7.7
100
Muscle tone poor, wasting
M
10
9
8'1
100
Marasmic
M
11
12
6.8
100
Marasmic
Jali
F
10
4
7.3
200
Muscle wasting
Jali
F
12
10
9.5
50
Jali
F
10
6
6-4
100
Poor condition, muscle wasting
Jali
M
12
9
9" 3
200
Muscle wasting, tone poor
Jali
F
15
12
4.5
200
Wasting of muscles
Kantonkunda
Keneba
2
2
Keneba Keneba
10
Keneba Jali
1
11
Muscle wasting, tone poor
B. Results in relation to village of residence Since both young children and adults resident in different villages were studied it is now proposed to examine results to determine the existence o f distinct patterns which might indicate epidemiological differences. (1) Children 0-4 years T h e distribution o f titres according to village o f residence is shown in Figure 9. A n t i b o d y titres for the children o f Keneba, Jali and Kantonkunda present a similar pattern, but those for M a n d u a r differ by showing a higher percentage in the low and intermediate ranges. Such a difference could easily arise i f in the child population o f M a n d u a r there was a greater p r o p o r t i o n o f young children than in the other 3 villages. I n Figure 10 the age group composition of the children is therefore shown, and it will be noted that the proportions o f children in M a n d u a r and Kantonkunda under 2 years o f age are very similar. Both Keneba and Jali populations contain higher proportions.
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IMMUNOFLUORESCENCE AND I M M U N E RESPONSE TO HYPERENDEMIC MALARIA
The reason for the high incidence of low antibody titres in Manduar, therefore, cannot be explained on the grounds of the age composition of its population. In Figures 11 and 12, showing respectively the incidence of aparasitaemia and impalpability of the spleen, it is apparent that, by these time-honoured criteria, the endemicity of malaria is lower in Manduar than in the other villages. This impression is further strengthened when the frequency of titres under 1:25 in the children of the 4 villages is considered; in Manduar 11 of 52 (21.15%) subjects occur in this range, while for the other 3 villages combined the incidence is 17 of 268 (6.34%). (2) Adults Of the 84 adults studied, 18 were residents of Manduar and 66 of either Jali or Keneba. The distribution of antibody titre and the incidence of parasitaemia in these individuals are shown in Figure 13. These results show that almost 38% of adults resident in Keneba or Jali possess antibody titres beyond the range demonstrable for Manduar residents. The mean log titres are 2.7782 and 3. 1082 for Manduar and other villages respectively. The difference is highly significant. Further, whereas no Manduar adult exhibited parasitaemia, one third of the Keneba-Jali sample did so. Thus, while it is recognized that the number of subiects observed in this investigation is small, there are consistent trends which indicate a different pattern of malarial endemicity in 1 of 4 closely related villages. Discussion
Since malaria is characteristically a dynamic infection, conclusions drawn from an isolated, cross-sectional study must be made with caution. With this qualification in mind, the following seem to be the salient results to have emerged from the present investigation. 1. In an area of hyperendemicity fluorescent antibody levels were found to be high in newborn infants, to fall in the weeks immediately following birth, to remain low for the remainder of the first year of life and thereafter to rise progressively throughout childhood into adult life. In these respects the findings confirm those obtained by VOLLER and BRAY (1962) in another hyperendemic area of West Africa, and conform to the classical concept of the acquisition of malarial immunity. 2. The rate of increase in antibody titre did not appear to be uniform. Rates were fastest in young children but slowed in adolescence and adult life. 3. The frequent failure to adduce evidence of recent malarial infection in young children exhibiting titres of less than 1:25 may constitute an important measure of the reliability and sensitivity of the technique employed. 4. The fall in parasite density noted to occur progressively and concomitantly with the rise in antibody titre in the age groups studied suggests a direct correlation between fluorescent antibody levels and the degree of effective immunity acquired. 5. Since neither density nor duration of parasitaemia alone appeared to be the dominant stimulus to antibody production, it is likely, as suggested by SINTON (1939) and MCGREGOR (1964) that the sum of these stimuli is more important than simply the magnitude of either. 6. No convincing evidence was gathered to indicate that, in young children, poor nutritional state adversely affected blood concentrations of antibody. Though KUVIN et al. (1962) have demonstrated the specificity of the fluorescent antibody test in malaria, the precise part played by assayable antibody in the mechanism
I. A. MCGREGOR, K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
413
of immunity remains unknown. The results of the present investigation suggest that it is intimately connected with, and consequently faithfully reflects, anfiplasmodial immunity. In the Gambia the first recognizable signs of developing immunity occur around the 3rd year of life and comprise a marked resistance to the pyrogenic and clinical effects of infection despite the persistence of moderately severe parasitaemia (MCGREGOR et al. 1956). The absence, therefore, of any sharp rise in fluorescent antibody titre around the 3rd year of life in the subjects studied may be significant, and may indicate that the substance measured is more concerned with antiparasitic activity. MCGREGOR and GILLES(1958, 1960) and GILLESand McGREGOR (1959, 1961) have traced the evolution of serum gamma globulin in Gambian Africans and have shown that levels of adult proportions are achieved by the 5th or 6th year of life. That fluorescent antibody titres continue to increase well into adult life is therefore noteworthy, for it may confirm the view (KuVlN et al., 1962; COHENand McGREGOR, 1963) that specific antiplasmodial antibody is not the sole or even the major factor responsible for the hypergammaglobulinaemia caused by repeated malarial infection. Consideration should be given to the possibility that the hypergammaglobulinaemia of early life, which develops in parallel with "clinical" immunity, arises from a spectrum of antibodies directed more against the products of parasite metabolism and erythrocyte breakdown than against the plasmodium itself. It is probable that the spleen functions as an important site of antibody synthesis. NORTONand WOLF (1949) and GRABARand CORVAZlER(1960) have reported a direct relationship between the weight of the spleen and its capacity for antibody production in chickens, and a similar association has been demonstrated in rabbits (MOUNTAIN, 1955). The observation that fluorescent antibody levels in African children rise as splenic size increases is therefore of interest and may confirm the view that in malaria the spleen, in addition to displaying intense phagocytic activity, plays an important role in specific antibody formation (BRUcE-CHWATT, 1956). Equally interesting is the fact that fluorescent antibody titres continued to rise throughout childhood and into adult life for, over this period, a significant reduction occurs in Africans in the ratio of spleen weight to body weight (BROcE-CHWATT, 1956). Consequently it would appear that in later life continuing increase in spleen size is not a prerequisite of increasing fluorescent antibody titre. Increase in antibody concentration of the blood over this period may occur from the development of other additional sites of antibody synthesis, from increased immunological competence on the part of the spleen, or from a progressive tendency of the organ to manufacture fewer antibodies in greater quantity. With regard to this last possibility it is conceivable that the advent of specific antiplasmodial antibody may decrease the need for such antibodies as may be associated with the acquisition of "clinical immunity". That malarial immunity may be attenuated in pregnancy is suspected (BRUCECHWATT, 1952; McGREGOR and SMITH, 1952; COHEN and MCGREGOR, 1963). In the present investigation, although no difference was detected in antibody titres of nonparasitized pregnant and non-pregnant women, substantial differences were noted between their parasitized counterparts. Although the numbers involved are small, such a difference invites speculation as to whether the pregnant female is less competent to contain the challenge of plasmodial infection, and suggests that immunofluorescence may well be used with profit in the study of immunological changes that attend pregnancy. There is today need for a technique capable of the accurate measurement of malaria endemicity (WORLD HEALTH ORGANIZATION REPORT, 1962; McGREGOR, 1965) and the data herein reported indicate that, in certain circumstances, fluorescent antibody assay may meet it admirably. The demonstration, for example, by simple cross-section study of the incidence of titres of less than 1:25 in children under 3 years of age, together
414
IMMUNOFLUORESCENCEAND IMMUNE RESPONSE TO HYPERENDEMID MALARIA
with the establishment of the titre range in adults, would constitute important information on both the frequency of malarial transmission and the degree of adult immunity achieved in any area. Such a view receives support from the contrast noted in findings between Manduar residents and the inhabitants of the other three villages. However, the fluorescent antibody test, as it is at present constituted, cannot be applied with ease to field conditions. The exacting requirements for storage of antigen and the constant need for vigilance in the preparation of glassware and reagents limit the test to competent technicians operating in a well equipped laboratory, while the accurate assessment of results obtained requires a detailed knowledge of the frequency of antimalarial chemotherapy in the area under survey. REFERENCES BANGnmVi, D. R. (1960). J. Physiol., 153, 265. BERTRAM,D. S., McGREGOR,I.A.&McFaDzEAN, J. A. (1958). Trans. R. Soc. Trop. Med. Hyg., 52, 135. BR.~mELL, F. W. R. (1958). Biol. Rev., 33, 488. BRUcE-Ct-rCCATT,L. J. (1952). Ann. trop. Med. Parasit., 46, 177. (1956). Bull. World Hlth. Org., 15, 513. COHEN, S. & HANSEN, J. D L. (1962). Clin. Sci., 23, 351. & MCGREGOR, I. A. (1963). Immunity to Protozoa. Oxford: BlackweU Scientific Publications. ,~ & CARRINGTON,S. (1961). Nature, Lond., 192, 733. COONS, A. H., CR~ECH, H. J. & JONES, R. N. (1941). Proc. Soc. exp. Biol. N.Y., 47~ 200. EDOZI~r, J. C. (1961). IV. Aft. reed. ~., 10, 304. GILLES, H. M. & McGREGOR, I. A. (1959). Ann. trop. Med. Parasit, 53~ 492. &~ (1961). Ibid., 55~ 463. G ~ A R , P. & CORVaZIER, P. (1960). Ciba Foundation Symposium on Cellular Aspects of Immunity. KUVIN, S. F., TOME, J. E., EVANS, C. B., COATNEY, G. R. & CONTACOS, P. G. (1962). Amer. J. trop. Med. Hyg., 11, 429. McGREGOR, I. A. (1964). Trans. R. Soc. trop. Med. Hyg., 58, 80. (1965). Ibid., 59~ 145. & GILLES, H. M. (1958). Proc. 6th Int. Congr. trop. Med. Malar., Lisbon, 7, 210. & (1960). Ann. trop. Med. Parasit., 54, 275. ~., - - , WALTERS, J. H., DAVIES, A. H. & PEARSON, F. A. (1956). Brit. med. J., 2~ 686. & SMITH, D. A. (1952). Trans. R. Soc. trop. Med. Hyg., 46~ 403. MOUNTAIN, I. M. (1955). J. Immunol., 74, 278. NORTON, S. & WOLFE, H. R. (1949). Anat. Rec., 105, 83. ROSS, R. (1910). The Prevention of Malaria. London: J. Murray. SINTON, J. A. (1939). J. Malar. Inst. India, 2, 191. VOLL~R, A. & BRAY, R. S. (1962). Proc. Soc. exp. Biol., N.Y.~ 110~ 907. WELLER, T. H. & CooNs, A. H. (1954). Ibid., 86, 789. World Health Organization Mimeographed Report of Third African Malaria Conference, Yaounde, 1962. WHO/Mal/376 (unpublished).
Vol. 59.
No. 4. July, 1965.
COMMUNICATIONS IMMUNOFLUORESCENCE A N D T H E M E A S U R E M E N T OF R E S P O N S E TO H Y P E R E N D E M I C MALARIA
IMMUNE
~Y I. A. McGREGOR AI~I)K. WILLIAMS The Medical Research Council Laboratories, Gambia, West Africa
A. VOLLER London School of Hygiene and Tropical Medicine AND
W. Z. BILLEWICZ* Medical Research Council, Obstetric Medicine Research Unit, Aberdeen Rates of splenomegaly and parasitaemia today form the basis for the measurement of malarial endemicity. Neither method is entirely satisfactory; enlargement of the spleen may be caused by other common diseases, and the incidence of parasitaemia alone can utterly faR' to present an adequate picture of the pattern of malaria in a population. For more than half a century it has been appreciated that the manifestations of malaria are largely governed by the degree of immunity possessed both by the individual and by the community (Ross, 1910). Any method, therefore, capable of measuring the immune state with even moderate accuracy would be of great importance in advancing our knowledge of malarial epidemiology, and would have an immediate application in the assessment of progress of eradication campaigns. Field studies made largely in Africa over the past 15 years have revealed a constant relationship between the rising concentration of serum gamma globulin and the acquisition of clinical immunity to plasmodial infection (MCGREGOR et al., 1956; MCGREGOR and GILLES, 1958 and 1960; GILLES and McGREGOR, 1959 and 1961; EDOZlEN, 1961) while, more recently, the passive transfer of malarial immunity in man has been demonstrated (COHEN, MCGREGOR and CARRINGTON,1961; COHEN and MCGREGOR, 1963). Over approximately the same period the serological specificity and potentialities of immunofluorescence have elsewhere been studied (CooNs, CREECH and JONES, 1941; WELLER and COONS, 1954). Lately these two fields of interest have converged and the measurement of specific antibody in the blood of malarious subjects by fluorescent immunological methods has been reported (KuVlN et al., 1962; VOLLERand BRAY, 1962). In this communication are reported the results of an attempt to measure by an immunofluorescent technique the levels of malarial antibody possessed by African subjects of different ages, resident in a hyperendemic environment. The investigations were made in January 1964 in 4 Mohammadan, Mandinka villages--Keneba, Jali, Manduar and Kantonkunda--situated in the West Kiang district of the Gambiaman area which has already been fully described (McGREGOR and SMITH, 1952; BERTRAM,McGREGOR and McFADZEAN, 1958). The study area is small and may * The authors gratefully acknowledge the financial support provided by the World Health Organization for this study. C
396
IMMUNOFLUORESCENCE AND IMMUNE RESPONSE TO HYPERENDEMIC MALARIA
be confined within a circle 5 miles in radius, centred upon Keneba. Within it the principal vectors are the fresh-water-breeding A. gambiae gambiae and its salt-water variant A. gambiae melas. Malaria, although hyperendemic, is not uniformly transmitted throughout the year. Mosquito density rises sharply before the onset of the rains in June, and initial populations are almost exclusively A. gambiae melas. As the rains progress, A. gambiae gambiae gradually replaces its variant and by August constitutes the dominant anopheline mosquito. Later, when the rains are about to cease, usually September-October, A. gambiae melas resumes dominance for a short time. While populations of A. gambiae gambiae seem evenly distributed throughout the 4 villages studied, those of A. gambiae melas appear to vary inversely with the distance of each village from the saline swamps. By January of each year the advent of hot and exceptionally dry weather drastically reduces anopheline density to low levels, which are then maintained until the following May. The transmission of malaria rises sharply in July and August and is maintained at a high level until November or December. Thereafter it declines and remains low for the rest of the year. Thus, when the investigations to be described were made, the human population had just emerged from the period of peak transmission and was entering the relatively non-malarious season.
Subjects and methods By a system of birth recording maintained for research purposes by the staff of the laboratories, the ages of all children under 5 years in the 4 villages were known with precision. Further, for the previous 2 years each available child in the study area had been examined at intervals of 3 months, when its general health, haematological indices, and malarial state had been assessed. Governed mainly by the unique facilities at hand, the primary objective was to determine the fluorescent antibody titre of each child under 5 years of age resident in the 4 villages, and to consider the results obtained against the available malariometric data. Accordingly 320 children were examined; the height and weight of each were recorded, health and nutritional states assessed, haemoglobin, packed cell volume and mean corpuscular haemoglobin concentration values determined, thick and thin blood films made for parasite identification and enumeration and, from fingerprick blood, a plasma sample was obtained for subsequent fluorescent antibody assay. A secondary objective was to determine fluorescent antibody levels in samples of older age groups, and to this end 16 children of 5-10 years, 26 of 11-16 years, and 84 adults were examined. The children aged 5-16 years were from Keneba only, since birth recording had been in operation longer there than in the other villages and since it was important to know the ages of subjects with accuracy. Of the adults, 18 were resident in Manduar, 17 in Jali, and 49 in Keneba; 44 were males and 40 females; of the latter 12 were pregnant. The older subjects were not medically examined; samples of blood for fluorescent antibody assay were taken by venepuncture and thick and thin blood films prepared from each subject. Thick blood films were stained by Field's method, and thin films after alcohol fixation, by Giemsa's. Parasites were enumerated against leucocytes and densities calculated by assuming a standard leucocyte count value of 8,000 per c.mm. 100 oil fields of each thick blood film were examined. Finger-prick blood samples were collected in heparinized capillary tubes, a minimum of 2 being taken from each subject. After sealing, the tubes were spun in a microcentrffuge for 5 minutes, the packed cell portion was broken off and the plasma made up in phosphate buffered saline to a dilution of 1:25 and stored at --20°C. until tested. As a check to the similarity of results obtained by finger-prick and venous blood collection, samples by both methods were taken from 20 adults. Comparison of antibody assay in these samples was satisfactory; in no
I. A. MCGREGOR~ K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
397
instance did results vary by more than one dilution--a variability not infrequently encountered in determining the end point of fuorescence in the serial dilution of any single serum sample. Thin blood films from patients exhibiting P. falciparum parasitaemia of not less than 30,000 trophozoites per c.mm. constituted the antigen. Films were air-dried over calcium chloride for 24 hours and then stored at --70°C. until required. They were then dehaemoglobinized in N/3 hydrochloric acid and, after washing with phosphate buffered physiological saline (pH 7" 2), layered with the appropriate dilution of plasma or serum under examination. After permitting interaction for 40 minutes, the film was again washed in buffered saline, dried and layered with fluorescein-labelled antihuman-globulin serum. 30 minutes later it was again washed in buffered saline. Finally, it was examined by fluorescence microscopy and the effective titre was taken to be that dilution at which fluorescence was last detected. The technique employed permitted titre assessment within broad groups only. Thus a titre reported as less than 1:25 embraces those from zero to 1:24, while those classified as 1:25 include dilutions in the range 1:25-1:49. For purposes of numerical analysis logarithmic transformation of titre has been used to normalize distributions and stabilize variances. Results
A. Results considered irrespective of village of residence Titre distribution within age groups is shown in Table I, while mean logarithmic titres for each age group are expressed graphically in Figure 1. These data clearly show that, after the early months of life, the antibody content of blood increases as age advances. In Table I, while a few relatively high values are recorded, the
MEAN
LOG TITRES (446
BY AGE GROUPS
subjects)
3.5"
30' . I
2.5' J
2.0
/
,../ 1.5, i
I
,s
|
¢"
(Mean ocje=33yeors)
Age
in y e a r s
Fro. 1.
398
IMMUNOFLUORESCENCE
AND
IMMUNE
RESPONSE
TO
HYPERENDEMIC
MALARIA
titres o f children aged less than 5 years are scattered fairly uniformly at the lower levels of the scale. For adults, values tend to be higher and there is a total absence o f low values. The relatively rapid decline in antibody levels in the months immediately after birth, probably indicating the decay of passively acquired immunity, is evident in Figure 1. Levels remain low until the end of the first year of life and thereafter increase throughout childhood into adult life. T h e rate of increase appears to be relatively fast in early childhood, and to become slower in older childhood and in adult life. TABLE I.
F.A. titres of the entire young child population (0-4 years) and of samples of older invidividuals resident in Keneba, Jali, Manduar or Kantonkunda.
Number Age ~ in Group Group <25 25 Months 0-
F.A. titre 50
Log titre
100 200 400 800 1600 3200 6400 Mean
S.D.
] 5
1
1]
2
2.6577 0"3432
1
I
1-
5
2
1
2-
11
4
2
3
--1"--~0620'3914
3-5
17
4
4
2
7
1.7451 0.4424
6-8
17
4
5
2
5
1
9-11
12
3
2--7
2
2
i] I
34
3
i 8-T
12
8
"
18-23
49
5
2.0660 0.4872
24-35
5
i 81 4- - - - V17m m11
3 .
m i62n i
2-1862 0.4154
36-47
57
2
48-59
51
Years 5-10
16
12-17
,
2
' 1.9955
0.4565
1.7274 0.4555
26
17
6 i
1-7809 0.5043 1
2-0189 0.4695
6
4
18
20
5
2
2.2438 0.4196
4
4
15
21
5
2
2.3237 0-3478
6 [ 4 I. 10 ! 8
2- 7405 0.2427
i
6
i
11-16
26
1
3
17 +
84
1
11
20
31 108 103
57
Total
446
28
44
4
2.9055 0.3090
26
20
3.0326 0.3544
45
24
In Figure 2 the individual titres of 25 infants aged less than 16 weeks are shown. Again, the rapid decline from high values in the first and second weeks o f life is apparent. Such a pattern supports the view that, in man, antibody transfer from mother to child is solely prenatal and transplacental (BRAMB~LL, 1958; BA~GHAM, 1960). It also suggests that the rate of decay of passively acquired immunity may be faster than is generally thought, and it should be recalled that, as some o f these infants had recently emerged from a season o f frequent malarial transmission, the rapid fall may have, in part, been the result of antibody utilization.
I. A, MCGREGOR~ K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
INDIVIDUAL TITRES OF 25 INFANTS
AGED LESS THAN
399
16 WEEKS
800700 600 500400300200IOO-
+- + ++++++ I L 2
3
5
4
;
-,
8
+
T
i
Io
,t
T
T
T
12
Age in weeks FIG. 2.
TITRE DISTRIBUTION WITHIN AGE GROUPS (.320 CHILDREN 5 YEARS OF AGE) 50
_ 0 - 3 months ( 2 5 )
30 t
2 4 - 3 5 months (62)
32.oo
30
t+oJ +oo
I0
'°1 I t I ~
4-11 months C42)
~.o+
~5o1 o -i
33-33
?.+,e l
I
I '.+' 3 6 - 4 7 months
(57)
23.8, 2r.,,,
30
0
2"p..t
u.~
- o WlO ", 0
.
50
1 2 - 2 3 months ( 8 3 )
I0 53
1~-77
3 5j
[3.s~ I I
48-59
C
months ( 5 1 ) 4118
u~ 3501 0
19 2 8
I0
9.64
22.89 7.23
3 6!
:~.41 '
~ ¢ ~ ° ° ~~
.? o ° o
0
I0 I
0 Titre FIG. 3.
~.q'~
0
0
0
400
IMMUNOFLUORESCENCE
AND IMMUNE
RESPONSE TO HYPERENDEMIC
MALARIA
In Figure 3 the distribution of titres of children under 5 years of age is shown. To counteract the effect of passively acquired antibody the infants are divided into two groups, 0-3 months and 4-11 months; thereafter older children are grouped by year of age. Again the general trend, manifest in a gradual shift into higher levels as age advances, suggests a slow consolidation of titre. The slowness of the change is obvious from the broad similarity of the histograms relating to the 3rd, 4th and 5th years of life, as well as from Figure 1. The specificity of the fluorescent antibody technique as applied to malaria has been demonstrated by KuvIN et al. (1962). It is necessary therefore that the titres under description should be considered in relation to the malarial experience of the subjects studied. In 28 of the 320 children under the age of 5 years antibody could not be demonstrated at a plasma dilution of 1:25. Table II comprises a list of these children and gives for each the age and malarial experience as judged by quarterly blood film examinations, wherever these were available, in the 2 preceding years. For 18 of the group no evidence of any malarial infection could be established; in the remaining 10, infection was definitely known to have occurred. Of these latter, only 3 harboured trophozoites in the circulating blood at examination in January 1964, and only 2 others were trophozoite-positive in November 1963. The table therefore indicates a strong correlation between low, even negligible, antibody concentrations, and the absence of recent malarial infection. In subject M.42 the year of apparent freedom from asexual parasitaemia which followed a year of ostensibly continuous infection may have been associated with the progressive decay of any immunity acquired. For patients J.52 and J.53 no explanation of the low titres observed can be offered. T~Lo. II.
The malarial experience, as judged by quarterly blood film examination, of children possessing F.A. titres < 1/25 Parasitaemia
Village Keneba K. 107
Age 1962 years months Jan. Apr.
1963 1964 July Nov. Jan. Apr. July Nov. Jan.
(Jan. 1964)
2
Neg. Neg.
65
5
Neg. Neg.
47
6
Neg. Neg.
43
8
Neg. Neg. Neg.
98
I
1
Neg. Neg. Neg.
2
71
5
57
6
36
11
64
N.S.N.S.N.S.
N.S. ! N . S . N . S . N . S . N . S .
Neg. Neg. 3
7 L
Neg.
Neg. Neg. Neg. Neg. Neg. Neg. I
io 4O
÷
+
÷ Gam.
+
+
Neg. Neg.
+
÷ Neg. Gam.
+
Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. i
401
I . A. M C G R E G O R ~ K. W I L L I A M S ~ A. V O L L E R A N D W . Z . B I L L E W I C Z
TABLE II~continued. Parasitaemia ~
ViHage
Age 1962 years months Jan. Apr.
r
1963 July Nov. Jan. ii Apr.
1964 July Nov.
Jan.
!
Manduar M. 37
Neg. Neg.
1
Neg. N e g .
26
Neg.
9
1
Neg.
Neg. Neg.
Neg. 4 Neg. Neg. Neg. I Gam.
24
1
1
Neg. Neg. Neg.
29
1
1
Neg
17
2
42
2
1
N.S.
44
2
1
Neg. Neg. Neg. Neg. Neg. N e g . Neg. Neg. Neg.
20
2
3
Neg. Neg. Neg.
49
2
5
N.S. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. i Neg. Neg. Neg. Neg. Neg. !
Gam. +
+
Gam. Gam. ] ÷ + ]Neg. Neg. Neg.
+
J
+
+
Neg. i Neg. Neg. Neg.
Jali J.
55
Neg. Neg.
13
Neg. Neg.
98
Neg. Neg.
29 52 53
r Neg. Neg. Neg.
8 r I
1
11
3
3
IKan tonkunda K.K. 28
Neg. N . S . N . S . N . S . N . S . N.S. N.S. N.S.
+
+ t
+
Neg.
+
+
+
+
+
Neg. Neg.
N.S. - - not examined. For the children of Keneba comprehensive medical records exist, spanning the 2 preceding years. In this period all medicines administered to sick children were carefully noted. These records show that treatment for acute P. falciparum malaria was afforded to subjects K.40 and K.71. It is of further interest to note that the father of subject K.64 was an employee of the Health Services of the Gambia Government and that he had long been suspected of intermittently administering antimalarials to his child. This point, however, could not be proved. For the children of the other villages no
402
IMMUNOFLUORESCENCE AND IMMUNE RESPONSE TO I-IYPERENDEMIC MALARIA
comprehensive medical records exist but it is known that access to antimalarials could only have been sporadic, if indeed it occurred at all. The titre distribution in all children under the age of 5 years according to the presence (241 subjects) or absence (79 subjects) of parasitaemia at the time of examination, is depicted in Figure 4. Again the correlation between patent infection and increased titre is apparent. TITRE DISTRIBUTION IN CHILDREN
AGED < 5 YEARS
(ACCORDING TO P A R A S I T A E M I A )
40-
Blood film posttlV¢
30-
=
20-
~ _"
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c
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I0-
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30"
Blood film negative
~ "/
at:) / 1 1
777-~
,/n~/Al--I~
~ l l Il l l Il
I
'25
25
50
I00
200
400
, 800
Titre
FIG. 4.
Figure 5 shows the titre distribution in gametocyte-positive (78) and gametocytenegative (163) infected children aged less than 5 years. It supplies little evidence to suggest that, in this age group at least, the presence or absence ofgametocytaemia is of any value as an index of immune state. An analysis of titre distribution according to density of parasitaemia for all children from 4 to 59 months of age is presented in Table Ill. It will be noted that titre distribution in the more heavily infected subjects tends to be restricted to the intermediate range, both high and very low titres being absent. However, this table gives no indication of relative rates of antibody synthesis and utilization, and it may well be that turnover rates in heavily infected individuals greatly exceeded those of their more lightly infected fellows.
I. A. MCGREGOR:, K. WILLIAMS, A. VOLLER AND W. Z. BILLEWICZ
TITRE DISTRIBUTION
IN M A L A R I O U S
403
CHILDREN
AGED 45 YEARS ~ACCORDING TO G A M E T O C Y T A E M I A ) T r o p h o z o i t e 4.v¢.
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,
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IOO
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800
Titr¢ F I G . 5.
In Table IV are shown the titre distributions according to the number of positive quarterly blood films recorded in 6 consecutive examinations for 153 children aged 24-59 months. Children under 24 months of age have been excluded since most were not of sufficient age to have been examined on 6 successive quarterly examinations and since these younger children tend to exhibit a titre pattern which is to a considerable extent age-confounded. It will be noted that the frequency of recorded parasitaemia does not appear to influence titre distribution, except in the 4 children possessing no record of infection in the preceding 18 months' period, all of whom show titres of less than 1:25. The results expressed in this table therefore strongly suggest that simple duration of parasitaemia is not the only, or possibly even the most important, factor in the progressive acquisition of immunity, and they should be considered in conjunction with those given in Table I I I . Together these suggest that neither density nor duration of parasitaemia is the single dominant factor in creating high concentration of antibody, and that consequently it is probably their combined effect that is important.
404
IMMUNOFLUORESCENCE AND I M M U N E RESPONSE TO HYPERENDEMIC MALARIA
TABLE III.
Titre distribution in children 4 to 59 months of age according to parasite density. F.A. Titre
No.
in 100 200 400 800 1600 group
<25
25
50
Blood film negative
21
16
4
8
4
2
Blood film positive; Parasites per c.mm. < 100
1
4
1
11
6
1
2
2
2
23
17
6
10
27
2
5
< 15,000
1
<20,000 > 20,000
< 1,000 <5,000 < i0,000
TABLE IV.
1
%
1 or2
%
3or4
%
5 6
S.D.
55
1.6108 0"5116
1
25
2"1570 0.4290
5
2
53
2.2887 0-3810
29
9
3
84
2.2979 0.3501
19
6
3
36
2.17120.3366
2
7
7
1
1
2
6
6
15
2"1833 0.3290
4
1
1
3r
9
F.A. titre <25
25
5O
100
Log titre
200
400
800
100.0 1
1
1
2
2
5 '9 3
2"9
11"8 4
8"8
6
9.
!7 11 "8
8
11 "8 6.3
1.5
Mean
S.D.
1-0969
--
100" 0
5"9
1
Total 4
4
o/ /o
%
Mean
Titre distribution according to number of positive quarterly blood films in six consecutive quarters (153 children aged 24-59 months).
No. of positive films None
Log Titre
3 41 "2
13
23"5
17 "6 1
4
38"2
5"9
11 "8
34 2.9
12 13 5 37.5 40"6 15 "6
17 3 1 7 10' 6 3045 •4 25 "8
2.2683 0.4181 99.9
32
2.3736 0"2491
100.0
2 4"5
17 2"3251 0.4917 100.1
66 3"0
2.2054 0- 3547 99.9
T h e range of parasite density within age groups is shown in Figure 6 for the 446 individuals studied, and the titre distribution for the same subjects is shown in similar fashion in Figure 7. T h e ability, as age advances, of the individual resident in hyperendemic circumstances to restrict parasite multiplication is clearly demonstrated i n Figure 6, and the concomitant progressive increase in antibody concentration is discernible in Figure 7. W h e n these figures are considered it should be remembered that, in the 0-4-years age
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406
IMMUNOFLUORESCENCE AND IMMUN'E RESPONSE TO HYPERENDEMIC MALARIA
group, are included members in the first few months of life who may not have experienced malarialinfection and who frequently possess high antibody levels inherited transplacentally. Figure 7 amply indicates how simple parasite rates fail to reflect the progress of the development of immunity. The similarity of such rates in the 3 young age groups obscures the considerable progress that is evident when parasite densities and antibody titres are considered. Viewed together, the findings illustrated in Figures 6 and 7 suggest that the antibody measured by the technique involved is intimately connected with antiparasitic immunity and may indeed be the specific antiplasmodial antibody. For many years the spleen rate has been regarded as an important malariometric datum, its magnitude in the age group 2-9 years being an index of malarial endemicity. In Figure 8 the titre range is contrasted of children 4-59 months of age with (226), and without (58), splenomegaly, while Table V relates titre to splenic size (Hackett's classification). Children in whom the accuracy of splenic palpation was dubious have been excluded. It will be noted that the frequency of low titres is considerably higher in children without splenic enlargement, and that increase in splenic size appears to be consistently associated with increase in antibody titre. The antibody titres in adults, classified by sex and parasite incidence and density, are shown in Table VI. TABLEV. F.A. titres of children 4 to 59 months of age according to splenic size (Hackett's classification) (Children whose spleens may have been imprecisely palpated are omitted). F.A. titre
Log titre
Group as No. percentage in of total group sample
--,[--
Size of spleen <25 25 0
14
12
1
4
6
2
5
13
1001200 400 800 Mean
50
3'191 5
1 1.8429 0"5363
58
20.42
2
2.0687 0"4526
43
15.14
46 i 10
4 2-2077 0'3896
153
53-87
2
1 2.3326 0.3596
25
8-80
2
2.4169 0-3298
5
1 "76
284
99" 99
i
57 .
.
.
3
7
4
3
TABLE VI.
1
8
1 4 , 12
18 .
S.D.
I
121
F.A. titres of adults according to parasitaemia at time of examination. F.A. titre
Group <25 25 5 0 Males Blood film neg.
Log titre in 100 200 400 800 1600 3200 group Mean S.D. No.
I
1
518
10
9
3
4
i
Blood film pos.
1
Females Blood film neg.
4
8 11
Blood film pos.
1
4
I 3
36
3"0290 0.3841 3" 1544 0"3116
1
26
2" 9403 0" 2978
2
14
3" 1222 0-3707
I. A. MCGREGOR~ K. WILLIAMS) A. VOLLER AND W. Z. BILLEWICZ
407
Though the group involved is small, the suggestion is apparent that women are represented in the higher titres to a lesser degree than men and, further, that patent parasitaemia is more frequent in them. Of the 40 female adults studied, 12 were pregnant, and of these 41.67% showed patent parasitaemia compared with 31.14% of the non-pregnant women. The titre distribution in these women is shown in Table VII.
TABLE VII.
F.A. titres of pregnant and non-pregnant women according to parasitaemia at time of examination. F.A. utre
Group <25 25 Pregnant Blood film neg.
50
Log titre in 100 200 400 800 1600 3200 group Mean S.D.
i
1
Blood film pos.
No.
3
2
3
2
1
7
2"9502 0"3830 2"8986 0' 1649
Non-pregnant Blood film neg.
3
4
10
2
Blood film pos.
1
1
1
4
2
19
2.9524 0.2714
9
3" 2464 0" 4014
Though mean log titres of pregnant and non-pregnant, unparasitized women are almost identical (2.9502, 2.9524 respectively), the mean log titre of pregnant, parasitized women (2. 8986) is substantially lower than that of parasitized non-pregnant women (3- 2464). With these small numbers, however, this difference, even although statistically significant, cannot be regarded as firmly established. The relationship of antibody titre to haematological indices (Hb, PCV, MCHC) was examined for children under 5 years of age. No pattern of improving haematological status with advancing antibody titre was observed. This result was not unexpected since in any child high antibody titre may well be the result of a recent severe attack of malaria. Thus anaemia and relatively high antibody titre must be expected to be simultaneously present in some proportion of young children. Among the 320 young children studied were 14 who were classified as being in poor nutritional state. The basis for such an assumption was a body weight well below that expected for the age of the individual, coupled with evidence of wasting and poor tone of the muscles. The relevant details are shown in Table V I I I and it will be noted that the antibody titres of these children provide no evidence to suspect that their nutritional state had materially affected their capacity to synthesize antibody. This finding accords with that of COHEN and HANSEN (1962), who found no evidence to indicate that the state of nutrition affected either the distribution or the turnover of gamma globulin in a group of children in South Africa. O f the 241 children aged less than 5 years known to be parasitized, 219 were infected with a single, 16 with two and 6 with three, species of plasmodia. Contrast of titres in these subjects revealed nothing which could be interpreted as indicating that multiple infections were associated with higher antibody levels than was infection with a single species.
408
IMMUNOFLUORESCENCEAND IMMUNE RESPONSE TO HYPERENDEMIC MALARIA TABLE V I I I . Village
Antibody titre in children with signs suggestive of malnutrition. Sex Age Years Months
Weight lb. oz.
Hb G.
Titre
Condition
i
M
15
15
9'0
100
Marasmic
Kantonkunda
M
28
1
9.7
100
Muscle tone poor, some wasting
Kantonkunda
M
14
12
12.4
100
Pronounced muscle wasting
Manduar
M
16
13
7.8
400
Pronounced muscle wasting
F
11
13
7.0
100
Marasmic
F
12
11½
11.7
25
Muscle wasting, tone poor
M
16
15
7.7
100
Muscle tone poor, wasting
M
10
9
8'1
100
Marasmic
M
11
12
6.8
100
Marasmic
Jali
F
10
4
7.3
200
Muscle wasting
Jali
F
12
10
9.5
50
Jali
F
10
6
6-4
100
Poor condition, muscle wasting
Jali
M
12
9
9" 3
200
Muscle wasting, tone poor
Jali
F
15
12
4.5
200
Wasting of muscles
Kantonkunda
Keneba
2
2
Keneba Keneba
10
Keneba Jali
1
11
Muscle wasting, tone poor
B. Results in relation to village of residence Since both young children and adults resident in different villages were studied it is now proposed to examine results to determine the existence o f distinct patterns which might indicate epidemiological differences. (1) Children 0-4 years T h e distribution o f titres according to village o f residence is shown in Figure 9. A n t i b o d y titres for the children o f Keneba, Jali and Kantonkunda present a similar pattern, but those for M a n d u a r differ by showing a higher percentage in the low and intermediate ranges. Such a difference could easily arise i f in the child population o f M a n d u a r there was a greater p r o p o r t i o n o f young children than in the other 3 villages. I n Figure 10 the age group composition of the children is therefore shown, and it will be noted that the proportions o f children in M a n d u a r and Kantonkunda under 2 years o f age are very similar. Both Keneba and Jali populations contain higher proportions.
I. A. MCGREGOR~
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IMMUNOFLUORESCENCE AND IMMUNE RESPONSE TO HYPERENDEMIC MALARIA
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IMMUNOFLUORESCENCE AND I M M U N E RESPONSE TO HYPERENDEMIC MALARIA
The reason for the high incidence of low antibody titres in Manduar, therefore, cannot be explained on the grounds of the age composition of its population. In Figures 11 and 12, showing respectively the incidence of aparasitaemia and impalpability of the spleen, it is apparent that, by these time-honoured criteria, the endemicity of malaria is lower in Manduar than in the other villages. This impression is further strengthened when the frequency of titres under 1:25 in the children of the 4 villages is considered; in Manduar 11 of 52 (21.15%) subjects occur in this range, while for the other 3 villages combined the incidence is 17 of 268 (6.34%). (2) Adults Of the 84 adults studied, 18 were residents of Manduar and 66 of either Jali or Keneba. The distribution of antibody titre and the incidence of parasitaemia in these individuals are shown in Figure 13. These results show that almost 38% of adults resident in Keneba or Jali possess antibody titres beyond the range demonstrable for Manduar residents. The mean log titres are 2.7782 and 3. 1082 for Manduar and other villages respectively. The difference is highly significant. Further, whereas no Manduar adult exhibited parasitaemia, one third of the Keneba-Jali sample did so. Thus, while it is recognized that the number of subiects observed in this investigation is small, there are consistent trends which indicate a different pattern of malarial endemicity in 1 of 4 closely related villages. Discussion
Since malaria is characteristically a dynamic infection, conclusions drawn from an isolated, cross-sectional study must be made with caution. With this qualification in mind, the following seem to be the salient results to have emerged from the present investigation. 1. In an area of hyperendemicity fluorescent antibody levels were found to be high in newborn infants, to fall in the weeks immediately following birth, to remain low for the remainder of the first year of life and thereafter to rise progressively throughout childhood into adult life. In these respects the findings confirm those obtained by VOLLER and BRAY (1962) in another hyperendemic area of West Africa, and conform to the classical concept of the acquisition of malarial immunity. 2. The rate of increase in antibody titre did not appear to be uniform. Rates were fastest in young children but slowed in adolescence and adult life. 3. The frequent failure to adduce evidence of recent malarial infection in young children exhibiting titres of less than 1:25 may constitute an important measure of the reliability and sensitivity of the technique employed. 4. The fall in parasite density noted to occur progressively and concomitantly with the rise in antibody titre in the age groups studied suggests a direct correlation between fluorescent antibody levels and the degree of effective immunity acquired. 5. Since neither density nor duration of parasitaemia alone appeared to be the dominant stimulus to antibody production, it is likely, as suggested by SINTON (1939) and MCGREGOR (1964) that the sum of these stimuli is more important than simply the magnitude of either. 6. No convincing evidence was gathered to indicate that, in young children, poor nutritional state adversely affected blood concentrations of antibody. Though KUVIN et al. (1962) have demonstrated the specificity of the fluorescent antibody test in malaria, the precise part played by assayable antibody in the mechanism
I. A. MCGREGOR, K. WILLIAMS~ A. VOLLER AND W. Z. BILLEWICZ
413
of immunity remains unknown. The results of the present investigation suggest that it is intimately connected with, and consequently faithfully reflects, anfiplasmodial immunity. In the Gambia the first recognizable signs of developing immunity occur around the 3rd year of life and comprise a marked resistance to the pyrogenic and clinical effects of infection despite the persistence of moderately severe parasitaemia (MCGREGOR et al. 1956). The absence, therefore, of any sharp rise in fluorescent antibody titre around the 3rd year of life in the subjects studied may be significant, and may indicate that the substance measured is more concerned with antiparasitic activity. MCGREGOR and GILLES(1958, 1960) and GILLESand McGREGOR (1959, 1961) have traced the evolution of serum gamma globulin in Gambian Africans and have shown that levels of adult proportions are achieved by the 5th or 6th year of life. That fluorescent antibody titres continue to increase well into adult life is therefore noteworthy, for it may confirm the view (KuVlN et al., 1962; COHENand McGREGOR, 1963) that specific antiplasmodial antibody is not the sole or even the major factor responsible for the hypergammaglobulinaemia caused by repeated malarial infection. Consideration should be given to the possibility that the hypergammaglobulinaemia of early life, which develops in parallel with "clinical" immunity, arises from a spectrum of antibodies directed more against the products of parasite metabolism and erythrocyte breakdown than against the plasmodium itself. It is probable that the spleen functions as an important site of antibody synthesis. NORTONand WOLF (1949) and GRABARand CORVAZlER(1960) have reported a direct relationship between the weight of the spleen and its capacity for antibody production in chickens, and a similar association has been demonstrated in rabbits (MOUNTAIN, 1955). The observation that fluorescent antibody levels in African children rise as splenic size increases is therefore of interest and may confirm the view that in malaria the spleen, in addition to displaying intense phagocytic activity, plays an important role in specific antibody formation (BRUcE-CHWATT, 1956). Equally interesting is the fact that fluorescent antibody titres continued to rise throughout childhood and into adult life for, over this period, a significant reduction occurs in Africans in the ratio of spleen weight to body weight (BROcE-CHWATT, 1956). Consequently it would appear that in later life continuing increase in spleen size is not a prerequisite of increasing fluorescent antibody titre. Increase in antibody concentration of the blood over this period may occur from the development of other additional sites of antibody synthesis, from increased immunological competence on the part of the spleen, or from a progressive tendency of the organ to manufacture fewer antibodies in greater quantity. With regard to this last possibility it is conceivable that the advent of specific antiplasmodial antibody may decrease the need for such antibodies as may be associated with the acquisition of "clinical immunity". That malarial immunity may be attenuated in pregnancy is suspected (BRUCECHWATT, 1952; McGREGOR and SMITH, 1952; COHEN and MCGREGOR, 1963). In the present investigation, although no difference was detected in antibody titres of nonparasitized pregnant and non-pregnant women, substantial differences were noted between their parasitized counterparts. Although the numbers involved are small, such a difference invites speculation as to whether the pregnant female is less competent to contain the challenge of plasmodial infection, and suggests that immunofluorescence may well be used with profit in the study of immunological changes that attend pregnancy. There is today need for a technique capable of the accurate measurement of malaria endemicity (WORLD HEALTH ORGANIZATION REPORT, 1962; McGREGOR, 1965) and the data herein reported indicate that, in certain circumstances, fluorescent antibody assay may meet it admirably. The demonstration, for example, by simple cross-section study of the incidence of titres of less than 1:25 in children under 3 years of age, together
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IMMUNOFLUORESCENCEAND IMMUNE RESPONSE TO HYPERENDEMID MALARIA
with the establishment of the titre range in adults, would constitute important information on both the frequency of malarial transmission and the degree of adult immunity achieved in any area. Such a view receives support from the contrast noted in findings between Manduar residents and the inhabitants of the other three villages. However, the fluorescent antibody test, as it is at present constituted, cannot be applied with ease to field conditions. The exacting requirements for storage of antigen and the constant need for vigilance in the preparation of glassware and reagents limit the test to competent technicians operating in a well equipped laboratory, while the accurate assessment of results obtained requires a detailed knowledge of the frequency of antimalarial chemotherapy in the area under survey. REFERENCES BANGnmVi, D. R. (1960). J. Physiol., 153, 265. BERTRAM,D. S., McGREGOR,I.A.&McFaDzEAN, J. A. (1958). Trans. R. Soc. Trop. Med. Hyg., 52, 135. BR.~mELL, F. W. R. (1958). Biol. Rev., 33, 488. BRUcE-Ct-rCCATT,L. J. (1952). Ann. trop. Med. Parasit., 46, 177. (1956). Bull. World Hlth. Org., 15, 513. COHEN, S. & HANSEN, J. D L. (1962). Clin. Sci., 23, 351. & MCGREGOR, I. A. (1963). Immunity to Protozoa. Oxford: BlackweU Scientific Publications. ,~ & CARRINGTON,S. (1961). Nature, Lond., 192, 733. COONS, A. H., CR~ECH, H. J. & JONES, R. N. (1941). Proc. Soc. exp. Biol. N.Y., 47~ 200. EDOZI~r, J. C. (1961). IV. Aft. reed. ~., 10, 304. GILLES, H. M. & McGREGOR, I. A. (1959). Ann. trop. Med. Parasit, 53~ 492. &~ (1961). Ibid., 55~ 463. G ~ A R , P. & CORVaZIER, P. (1960). Ciba Foundation Symposium on Cellular Aspects of Immunity. KUVIN, S. F., TOME, J. E., EVANS, C. B., COATNEY, G. R. & CONTACOS, P. G. (1962). Amer. J. trop. Med. Hyg., 11, 429. McGREGOR, I. A. (1964). Trans. R. Soc. trop. Med. Hyg., 58, 80. (1965). Ibid., 59~ 145. & GILLES, H. M. (1958). Proc. 6th Int. Congr. trop. Med. Malar., Lisbon, 7, 210. & (1960). Ann. trop. Med. Parasit., 54, 275. ~., - - , WALTERS, J. H., DAVIES, A. H. & PEARSON, F. A. (1956). Brit. med. J., 2~ 686. & SMITH, D. A. (1952). Trans. R. Soc. trop. Med. Hyg., 46~ 403. MOUNTAIN, I. M. (1955). J. Immunol., 74, 278. NORTON, S. & WOLFE, H. R. (1949). Anat. Rec., 105, 83. ROSS, R. (1910). The Prevention of Malaria. London: J. Murray. SINTON, J. A. (1939). J. Malar. Inst. India, 2, 191. VOLL~R, A. & BRAY, R. S. (1962). Proc. Soc. exp. Biol., N.Y.~ 110~ 907. WELLER, T. H. & CooNs, A. H. (1954). Ibid., 86, 789. World Health Organization Mimeographed Report of Third African Malaria Conference, Yaounde, 1962. WHO/Mal/376 (unpublished).