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Further studies on the yellow fever haemagglutination test
344 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 50.
No. 4.
July, 1956.
COMMUNICATIONS FURTHER
STUDIES
ON THE YELLOW
FEVER HAEMAGGLUTINATION
TEST
BY
J. S. P O R T E R F I E L D , M.D.e National Institute for Medical Research, London. (Seconded to the West African Council for Medical Research Laboratories, Lagos).
T h e a p p l i c a t i o n o f t h e h a e m a g g l u t i n a t i o n - i n h i b i t i o n t e s t ( H . A . I . test) to t h e d i a g n o s i s of y e l l o w fever in m a n was r e p o r t e d in an earlier c o m m u n i c a t i o n (PORTERFIELD, 1954). F u r t h e r s t u d i e s on t h e specificity o f t h e r e a c t i o n c o n f i r m e d t h e f i n d i n g s of CASALS a n d BROWN (1954) t h a t t h e r e was e x t e n s i v e serological o v e r l a p p i n g b e t w e e n y e l l o w fever a n d c e r t a i n o t h e r a r t h r o p o d - b o r n e v i r u s e s w h i c h t h e y classified as G r o u p B. H o w e v e r , t h e r e is e v i d e n c e t h a t at least in t h e case of m o n k e y s i n f e c t e d w i t h U g a n d a S, Zika, W e s t N i l e a n d D e n g u e A viruses, t h e cross r e a c t i o n s are a t r a n s i e n t p h e n o m e n o n w h i c h d i s a p p e a r w i t h i n 3 m o n t h s of infection (PORTERFIELD, 1955). S i n c e t h e g r e a t e s t usefulness of t h e y e l l o w fever H . A . I . test w o u l d b e as a s u b s t i t u t e for t h e classical m o u s e p r o t e c t i o n t e s t in t h e e x a m i n a t i o n of h u m a n sera, it s e e m e d d e s i r a b l e to e x t e n d t h e i n f o r m a t i o n a l r e a d y p r e s e n t e d b y e x a m i n i n g a l a r g e r n u m b e r of sera c o l l e c t e d f r o m several different areas. I n t h e c o u r s e of a visit to t h e G o l d C o a s t in O c t o b e r a n d N o v e m b e r , 1954, t h e o p p o r t u n i t y arose to collect a p p r o x i m a t e l y 400 h u m a n sera f r o m seven w i d e l y s c a t t e r e d areas. T h r e e h u n d r e d a n d s e v e n t y - f o u r sera h a v e n o w b e e n e x a m i n e d b y b o t h H . A . I . a n d m o u s e p r o t e c t i o n tests a n d t h e r e s u l t s of t h e s e are h e r e p r e s e n t e d . MATERIALS AND METHODS These were as previously described (PORTERFIELD,1954) with the following modifications : Antigen. This was stored as a freeze dried preparation of infected monkey serum extract. Under these conditions full potency was retained for at least 12 months. Antibody extract. T h e Seitz filtration technique of CASALS and BROWN (1954), with minor modifications, was found to be superior to the acetone extraction procedure. Blood cells. A photo-electric colorimeter was used to standardize cells from 1-day-old chicks to approximately 0.5 per cent. by volume. Diluent. Phosphate buffered saline p H 7.0 and M/10 was used as diluent. Mouse protection test. Survival ratio criteria (SAWYERand LLOYD,1931) were used instead of average survival times. As a check upon the reliability of the H.A.I. tests all extracts were tested twice and the mean of the two titres used in the analysis. ~ M y thanks are due to the Chief Medical Officer to the Gold Coast Government for his co-operation in this survey ; to the Medical Officers and others in the areas visited for their valued assistance; to the Director and staff of the West African Council for Medical Research Laboratories for facilities granted there; and to Miss Marjorie J. Mnssett, National Institute for Medical Research, for f i e statistical analysis.
J. s. PORTERFIELD
345
F i g u r e 1 shows t h e geographical locations of t h e seven areas visited. T h e s e areas were selected so as to r e p r e s e n t different types of c o u n t r y r a n g i n g f r o m t h i c k forest in t h e s o u t h to o p e n s a v a n n a h in t h e n o r t h . A n a t t e m p t was m a d e to collect sera f r o m r e p r e s e n t a t i v e samples in different age-groups, a n d f r o m e q u a l n u m b e r s of males a n d females. D o n o r s were u n s e l e c t e d w i t h r e g a r d to past history of illnesses, except in t h e K p a n d a e area, details of w h i c h are g i v e n later. T h e results o b t a i n e d b y u s i n g each of the two tests h a v e b e e n a r r a n g e d a c c o r d i n g to the areas f r o m w h i c h the sera were o b t a i n e d a n d t h e age-groups of t h e subjects ( T a b l e I). T a b l e I I shows t h e n u m b e r s o f s e r a in each area w h i c h gave t h e same (or different) results b y t h e P . T . a n d H . A . l , tests. T h e values of Z2 given in this table are calculated a c c o r d i n g to t h e m e t h o d d e s c r i b e d b y DENTON a n d BEECHER (1949) for use w h e n t h e r e is correlation b e t w e e n t h e two g r o u p s u n d e r c o m p a r i s o n . T h e s e values m e a s u r e t h e significance of t h e n u m b e r of d i s a g r e e m e n t s w i t h i n each area. T h u s a n o n - s i g n i f i c a n t X2 (groups I, II, I I I a n d V) indicates good a g r e e m e n t b e t w e e n t h e two tests a n d a significant Z2 p o o r agreement. RESULTS
Area I. Old Tafo. T h i r t y - o n e sera i,vere tested from Tafo which lies in the forest belt about 75 miles north of Accra and is a centre of cocoa farming. Since there is considerable movement of population into the cocoa regions, only persons who had spent their entire lives in and around Taro were included in the series. Four sera were protection-test positive and one doubtful. Only three sera were positive in H.A.I. tests, two of these being also P.T. positive. Area H.
FIG. 1. M a p of t h e G o l d Coast, s h o w i n g locations of t h e seven areas in w h i c h b l o o d s p e c i m e n s were taken. H a t c h e d p o r t i o n indicates a p p r o x i m a t e e x t e n t of forest zone.
Apinamang, near Akwatia.
Dr. J. J. MESSENT, Medical Officer at the C.A.S.T. Hospital, Akwatia, reported that cases of jaundice believed to be due to yellow fever are not uncommonly seen there, and protection tests on sera submitted by him to the Virus Unit at Yaba had revealed a f~irly high proportion of positives. Since, however, many of the employees of the mining concern are drawn from other parts of the country, it was felt that a survey of a village in the bush would give a better idea of the state of yellow fever iramunity in the local population. Accordingly, 42 sera were collected at Apinamang, a village with a population of about 2,000 situated in fairly thick bush about 12 miles from Akwatia. Eight sera were positive in protection tests ; five sera were positive by H.A.I. test, all of them being also P.T. positive.
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TABLE II.
Agreement between P.T. and H.A.I. tests in seven different areas. II
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43
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562 158
263
43 200
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1
Punpuano Village, near Kintampo.
K i n t a m p o is a town lying in hilly country at about 1,200 feet in an area where true forest is ending and guinea savannah type of country is beginning. Little is known of the incidence of yellow fever in the area, the last known case being that of a European who died of yellow fever in 1931. An outbreak of fever associated with jaundice was seen by Dr. B. B. WADDY and the staff of Medical Field Units in and around K i n t a m p o in M a y and June, 1954. H e considered that this was probably a virus infection, but thought yellow fever unlikely because of the mild nature of the illnesses observed. T h e village of Punpuano, about eight miles f r o m Kintampo, was suggested by Dr. WADDY and 61 samples of s e r u m were collected well distributed over the different age-groups. T h e r e were no protection-test positives in the under 5 age-group ; four out of 10 in the 5-9 age-group were positive, with a steady increase in the n u m b e r of positives in the older age-groups. H.A.I. tests gave substantially similar results with agreement between the two tests in 53 cases as against eight disagreements. Detailed results of the two tests and the age distribution of the positives are shown in Fig. 2. T h e relatively high proportion of protective sera, especially in the group aged 5-9, indicates that yellow fever is probably endemic in the area ; without a detailed examination of those involved it is impossible to determine whether or not the illness prevalent in M a y and June was yellow fever, although this seems not improbable on the protection test results.
Area IV.
Kpandae.
Cases of jaundice were reported f r o m K p a n d a e in September, 1954. T h e area was visited b y the Medical Officer from Yendi, who reported that the condition was almost certainly not yellow fever, and p u t forward the diagnosis of infective hepatitis. T h e town was visited in c o m p a n y with the Medical Officer and further cases of jaundice were seen. T h e illness appeared to start with a sharp febrile episode, followed in about 5 days by the onset of jaundice, which was often intense and persisted for 3 or 4 weeks. T h e cases seen were all in adults over the age of 20 and were equally c o m m o n in m e n and women. Convalescents
348
H A E M A G G L U T I N A T I O N TEST I N Y E L L O W FEVER
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J. s. P O R T E R F I E L D
349
and apparently healthy persons in roughly equivalent age-groups were bled, together with a number of children who gave no history of jaundice. A total of 41 sera was collected. The serum of one child out of six in the 5-9 age-group, and one out of six in the 10-14 age-group, were positive in protection tests, although no cases of jaundice were recognized amongst the children. In the 20-29 age-group seven out of 14 sera were P.T. positive, five out of seven in the 30-39 age-group, and all sera from persons over the age of 40 were positive. There were five specimens from persons who had been jaundiced for at least 10 days which gave negative mouse protection tests, thus excluding the diagnosis of yellow fever in those persons, and the P.T. positive sera were distributed almost equally between jaundiced and apparently healthy individuals. It thus appears that the jaundice was not due to yellow fever. Comparison of the H.A.I. and P.T. results shows 14 disagreements out of 41 sera, 11 of these being H.A.I. positive but P.T. negative.
Area V. Tamale. Tamale, the administrative centre of the Northern Territories, is a medium-sized market town situated on a low hill in the centre of country ranging from guinea savannah to true savannah. The last known outbreak of yellow fever was in 1931 when six Europeans died, the infection being apparently introduced from Pong Tamale 20 miles further north. The villages of Chogu and Yohni were visited and 96 sera were collected. These were examined in separate protection tests in which the levels of yellow fever virus employed were respectively 562 and 158 LDS0. The results in the two villages were very similar, and have been combined in the analysis. The youngest persons having positive sera were two females aged 25 in Chogu village, out of a total of 15 examined in that age-group. Three out of 14 sera were positive by P.T. in the 30-39 age-group, and 14 out of 15 in the group aged 40 and above. These findings are in agreement with the known history of yellow fever and suggest that there have been no further introductions of the disease since 1931. Two sera from children gave positive H.A.I. tests ; one was from a femal e aged 2 whose serum gave titres of 10 and 12.5 in two tests ; the other was from a boy of 14 which gave titres of 12.5 and 20. Otherwise the H.A.I. results are in close agreement with those obtained by P.T. Detailed results are presented in Fig. 3. Area VI. Pong Tamale. Thirty-nine specimens were collected in the native village of Pong Tamale. Apart from one serum from a'girl of 10, all specimens from persons under the age of 27 gave negative protection test results, whilst of sera from 16 persons over that age only one was negative and one doubtful. All 16 specimens from persons over 27 were also H.A.I. positive, as was that from the 10-year-old girl. The 11 specimens which were H.A.I. positive, but P.T. negative, were mostly from children, the youngest being aged 3½. Area VII. Bolgatanga. The two villages of Bongo and Yorogo, less than 10 miles south of the northern boundary of the Gold Coast, were selected as more suitable for survey purposes than the town of Bolgatanga itself. Both villages are in open, rocky country, with wide areas of grassland and only occasional stunted trees or bushes. Enquiry produced no history of yellow fever in Bolgatanga ; the nearest recorded cases of yellow fever were in a Syrian who died at Wale
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FIG. 4. A r e a V I I . Bolgatanga. I n each of F i g u r e s 2, 3 a n d 4, t h e lower section shows t h e results of b o t h P . T . a n d H . A . I . tests o n i n d i v i d u a l sera a r r a n g e d a c c o r d i n g to age-groups. I n each a g e - g r o u p t h e left c o l u m n r e p r e s e n t s P . T . results a n d t h e r i g h t c o l u m n H . A . I . results. A n o p e n s q u a r e indicates a negative result ; a closed square, a positive result ; a n d a h a t c h e d square, a d o u b t f u l result. T h e central section shows t h e p e r c e n t a g e of positive tests in each age-group. Solid l i n e - H . A . I . test, d o t t e d l i n e - - P . T . T h e u p p e r section shows t h e g e o m e t r i c m e a n s of t h e titres of sera giving positive H . A . I . tests.
352
I-IAEMAGGLUTINATIONTEST IN YELLOWFEVER
Wale, 35 miles south of Bolgatanga, in 1931, and two Europeans who died in Lawra and T u m u in 1936 and 1938. These two towns are on the same latitude as Bolgatanga but 140 and 80 miles west, respectively. Sixty-four sera were collected in all, and the results from the two villages have been combined. T h e youngest subjects from whom definitely positive sera were obtained were four young men of 25 ; above that age, sera from all but three out of 26 persons gave positive protection tests. An interesting feature of the survey in this area was the high proportion of doubtful results, not less than 11 out of 64 in the original tests. T h e doubtfuls are not due to the low dose of virus in one test (43 LD50), since they occur about equally in the two villages in separate runs of protection tests. W h e n nine of these were re-tested, one gave a clear positive result, five gave negative results and three remained doubtful in a test employing 186 L D 5 0 of yellow fever virus. Fig. 4 gives detailed results of H.A.I. and P.T. results and indicates the age distribution of the specimens. It will be seen that H.A.I. positive sera occur in the youngest age-group tested and increase in frequency with age, so that only one specimen from a person over the age of 20 is H.A.I. negative. HAEMAGGLUTINATION I N H I B I T I O N TITRES
T h e results so far presented have been based upon a simple positive or negative reading of the H.A.I. test, depending upon the presence or absence respectively of inhibitory substance in a 1 : 10 dilution of serum extract. In practice all sera were first tested in dilutions of up to 1 : 40 since the presence o f a prozone phenomenon indicated that a screening test on sera at 1 : 10 dilution only might miss a proportion of positives. Subsequently all positive extracts were re-tested in further dilutions to reach an endpoint. Such tests were repeated a second time and the geometric mean of the two titres was taken as the final result. T h e reproducibility of the test was analyzed by tabulating the differences between the two observations. Of 125 pairs of observations, the mean difference was 0.03, and the standard deviation 0.90, where 1.0 equals one twofold dilution. Since the haemagglutinating dose varied from 10 to 20 in different tests, and the titre of a serum bears a linear relationship to the test dose, the reproducibility of the result is reasonably good. T h e geometric means of the H.A.I. titres of sera from the different areas are included in T a b l e I. In Areas I, II, III, IV, and V the titres are all generally low and show no particular pattern. In Areas VI and VII, in the Northern Territories, the sera show a steady increase in H.A.I. titres with advancing age. DISCUSSION
In Areas I, II, I I I and V, the two tests show an agreement which is statistically highly significant. Taking the results Of these four areas together, the over-all agreement between the two tests is 91 per cent. of 230 sera examined. In these areas then, the yellow fever H.A.I. test appears to give a fairly reliable indication of the immunity state of the" population as determined by the mouse protection test, and cross immunity due to the presence of other viruses does not complicate the interpretation of the results. In Area IV, Kpandae, the agreement between the two tests is less significant. At first it appeared possible that the rather high level of 900 L D 5 0 of virus used in the protection test might have explained some of the H.A.I. positive, but P.T. negative, findings. However, when certain of these were re-tested with a lower L D 5 0 they remained negative. T h e
J. s. PORTERFIELD
353
Kpandae sera are not, however, a random selection since they include specimens from persons bled in convalescence from an undiagnosed illness. It is possible that the agent responsible for the jaundice may have caused the excess of H.A.I. positive sera found in this group ; the evidence on this point is not conclusive. Whatever the explanation, it is clear that with this particular set of sera, collected at this particular time, the H.A.I. test is an unreliable measure of yellow fever immunity as determined by mouse protection tests. At Pong Tamale the agreement is poor, and at Bolgatanga only 53 per cent. of all the sera give the same result by both tests. It has already been noted that these two areas differ from the rest in that they show increasing H.A.I. titres with advancing age. The absolute levels of H.A.I. titres in these areas are higher than would be expected if there had been no yellow fever for about 20 years, as the P.T. results seem to suggest. The most reasonable explanation for these two observations would seem to be the presence in the north of the Gold Coast of an agent or agents serologically related to yellow fever virus which produces antibody cross reacting with yellow fever antigen in H.A.I. tests. So little is known of the distribution of the various tropical viruses throughout West Africa, that it is difficult without testing sera against all the known possible agents, to determine which might be responsible. Further studies on this problem are proceeding. SUMMARY
(1) Throughout the southern regions of the Gold Coast, agreement between yellow fever P.T. and H.A.I. results is sufficiently close for the latter to be used as an indication of immunity levels in the population. Near the northern boundaries of the Gold Coast this relationship does not hold, and reliance on H.A.I. results would give very misleading results. (2) To guard against possible confusion due to the presence of serologically related viruses, it is suggested that every fifth serum from adults, and possibly all sera from children, showing a positive H.A.I. result, should be checked by yellow fever protection tests. Similar or more frequent checks should be carried out in areas where there is evidence of recent virus infection. REFERENCES CASALS, J. & BROWN,L. V. (1954). J. exp. Med., 99, 429. DENTON, J. E. • BELCHER,H. K. (1949). J. Amer. reed. Ass., 141, 1051. PORTERFIELD, J. S. (1954). Trans. R. Soc. trop. Med. Hyg., 48, 261. (1955). Proceedings VIIth International Congress of Comparative Pathology. Lausanne, 1955. SAWYER, W. A. ~x;LLOYD,W. (1931). J. exp. Med., 54, 533.
-
-
Vol. 50.
No. 4.
July, 1956.
COMMUNICATIONS FURTHER
STUDIES
ON THE YELLOW
FEVER HAEMAGGLUTINATION
TEST
BY
J. S. P O R T E R F I E L D , M.D.e National Institute for Medical Research, London. (Seconded to the West African Council for Medical Research Laboratories, Lagos).
T h e a p p l i c a t i o n o f t h e h a e m a g g l u t i n a t i o n - i n h i b i t i o n t e s t ( H . A . I . test) to t h e d i a g n o s i s of y e l l o w fever in m a n was r e p o r t e d in an earlier c o m m u n i c a t i o n (PORTERFIELD, 1954). F u r t h e r s t u d i e s on t h e specificity o f t h e r e a c t i o n c o n f i r m e d t h e f i n d i n g s of CASALS a n d BROWN (1954) t h a t t h e r e was e x t e n s i v e serological o v e r l a p p i n g b e t w e e n y e l l o w fever a n d c e r t a i n o t h e r a r t h r o p o d - b o r n e v i r u s e s w h i c h t h e y classified as G r o u p B. H o w e v e r , t h e r e is e v i d e n c e t h a t at least in t h e case of m o n k e y s i n f e c t e d w i t h U g a n d a S, Zika, W e s t N i l e a n d D e n g u e A viruses, t h e cross r e a c t i o n s are a t r a n s i e n t p h e n o m e n o n w h i c h d i s a p p e a r w i t h i n 3 m o n t h s of infection (PORTERFIELD, 1955). S i n c e t h e g r e a t e s t usefulness of t h e y e l l o w fever H . A . I . test w o u l d b e as a s u b s t i t u t e for t h e classical m o u s e p r o t e c t i o n t e s t in t h e e x a m i n a t i o n of h u m a n sera, it s e e m e d d e s i r a b l e to e x t e n d t h e i n f o r m a t i o n a l r e a d y p r e s e n t e d b y e x a m i n i n g a l a r g e r n u m b e r of sera c o l l e c t e d f r o m several different areas. I n t h e c o u r s e of a visit to t h e G o l d C o a s t in O c t o b e r a n d N o v e m b e r , 1954, t h e o p p o r t u n i t y arose to collect a p p r o x i m a t e l y 400 h u m a n sera f r o m seven w i d e l y s c a t t e r e d areas. T h r e e h u n d r e d a n d s e v e n t y - f o u r sera h a v e n o w b e e n e x a m i n e d b y b o t h H . A . I . a n d m o u s e p r o t e c t i o n tests a n d t h e r e s u l t s of t h e s e are h e r e p r e s e n t e d . MATERIALS AND METHODS These were as previously described (PORTERFIELD,1954) with the following modifications : Antigen. This was stored as a freeze dried preparation of infected monkey serum extract. Under these conditions full potency was retained for at least 12 months. Antibody extract. T h e Seitz filtration technique of CASALS and BROWN (1954), with minor modifications, was found to be superior to the acetone extraction procedure. Blood cells. A photo-electric colorimeter was used to standardize cells from 1-day-old chicks to approximately 0.5 per cent. by volume. Diluent. Phosphate buffered saline p H 7.0 and M/10 was used as diluent. Mouse protection test. Survival ratio criteria (SAWYERand LLOYD,1931) were used instead of average survival times. As a check upon the reliability of the H.A.I. tests all extracts were tested twice and the mean of the two titres used in the analysis. ~ M y thanks are due to the Chief Medical Officer to the Gold Coast Government for his co-operation in this survey ; to the Medical Officers and others in the areas visited for their valued assistance; to the Director and staff of the West African Council for Medical Research Laboratories for facilities granted there; and to Miss Marjorie J. Mnssett, National Institute for Medical Research, for f i e statistical analysis.
J. s. PORTERFIELD
345
F i g u r e 1 shows t h e geographical locations of t h e seven areas visited. T h e s e areas were selected so as to r e p r e s e n t different types of c o u n t r y r a n g i n g f r o m t h i c k forest in t h e s o u t h to o p e n s a v a n n a h in t h e n o r t h . A n a t t e m p t was m a d e to collect sera f r o m r e p r e s e n t a t i v e samples in different age-groups, a n d f r o m e q u a l n u m b e r s of males a n d females. D o n o r s were u n s e l e c t e d w i t h r e g a r d to past history of illnesses, except in t h e K p a n d a e area, details of w h i c h are g i v e n later. T h e results o b t a i n e d b y u s i n g each of the two tests h a v e b e e n a r r a n g e d a c c o r d i n g to the areas f r o m w h i c h the sera were o b t a i n e d a n d t h e age-groups of t h e subjects ( T a b l e I). T a b l e I I shows t h e n u m b e r s o f s e r a in each area w h i c h gave t h e same (or different) results b y t h e P . T . a n d H . A . l , tests. T h e values of Z2 given in this table are calculated a c c o r d i n g to t h e m e t h o d d e s c r i b e d b y DENTON a n d BEECHER (1949) for use w h e n t h e r e is correlation b e t w e e n t h e two g r o u p s u n d e r c o m p a r i s o n . T h e s e values m e a s u r e t h e significance of t h e n u m b e r of d i s a g r e e m e n t s w i t h i n each area. T h u s a n o n - s i g n i f i c a n t X2 (groups I, II, I I I a n d V) indicates good a g r e e m e n t b e t w e e n t h e two tests a n d a significant Z2 p o o r agreement. RESULTS
Area I. Old Tafo. T h i r t y - o n e sera i,vere tested from Tafo which lies in the forest belt about 75 miles north of Accra and is a centre of cocoa farming. Since there is considerable movement of population into the cocoa regions, only persons who had spent their entire lives in and around Taro were included in the series. Four sera were protection-test positive and one doubtful. Only three sera were positive in H.A.I. tests, two of these being also P.T. positive. Area H.
FIG. 1. M a p of t h e G o l d Coast, s h o w i n g locations of t h e seven areas in w h i c h b l o o d s p e c i m e n s were taken. H a t c h e d p o r t i o n indicates a p p r o x i m a t e e x t e n t of forest zone.
Apinamang, near Akwatia.
Dr. J. J. MESSENT, Medical Officer at the C.A.S.T. Hospital, Akwatia, reported that cases of jaundice believed to be due to yellow fever are not uncommonly seen there, and protection tests on sera submitted by him to the Virus Unit at Yaba had revealed a f~irly high proportion of positives. Since, however, many of the employees of the mining concern are drawn from other parts of the country, it was felt that a survey of a village in the bush would give a better idea of the state of yellow fever iramunity in the local population. Accordingly, 42 sera were collected at Apinamang, a village with a population of about 2,000 situated in fairly thick bush about 12 miles from Akwatia. Eight sera were positive in protection tests ; five sera were positive by H.A.I. test, all of them being also P.T. positive.
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Punpuano Village, near Kintampo.
K i n t a m p o is a town lying in hilly country at about 1,200 feet in an area where true forest is ending and guinea savannah type of country is beginning. Little is known of the incidence of yellow fever in the area, the last known case being that of a European who died of yellow fever in 1931. An outbreak of fever associated with jaundice was seen by Dr. B. B. WADDY and the staff of Medical Field Units in and around K i n t a m p o in M a y and June, 1954. H e considered that this was probably a virus infection, but thought yellow fever unlikely because of the mild nature of the illnesses observed. T h e village of Punpuano, about eight miles f r o m Kintampo, was suggested by Dr. WADDY and 61 samples of s e r u m were collected well distributed over the different age-groups. T h e r e were no protection-test positives in the under 5 age-group ; four out of 10 in the 5-9 age-group were positive, with a steady increase in the n u m b e r of positives in the older age-groups. H.A.I. tests gave substantially similar results with agreement between the two tests in 53 cases as against eight disagreements. Detailed results of the two tests and the age distribution of the positives are shown in Fig. 2. T h e relatively high proportion of protective sera, especially in the group aged 5-9, indicates that yellow fever is probably endemic in the area ; without a detailed examination of those involved it is impossible to determine whether or not the illness prevalent in M a y and June was yellow fever, although this seems not improbable on the protection test results.
Area IV.
Kpandae.
Cases of jaundice were reported f r o m K p a n d a e in September, 1954. T h e area was visited b y the Medical Officer from Yendi, who reported that the condition was almost certainly not yellow fever, and p u t forward the diagnosis of infective hepatitis. T h e town was visited in c o m p a n y with the Medical Officer and further cases of jaundice were seen. T h e illness appeared to start with a sharp febrile episode, followed in about 5 days by the onset of jaundice, which was often intense and persisted for 3 or 4 weeks. T h e cases seen were all in adults over the age of 20 and were equally c o m m o n in m e n and women. Convalescents
348
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and apparently healthy persons in roughly equivalent age-groups were bled, together with a number of children who gave no history of jaundice. A total of 41 sera was collected. The serum of one child out of six in the 5-9 age-group, and one out of six in the 10-14 age-group, were positive in protection tests, although no cases of jaundice were recognized amongst the children. In the 20-29 age-group seven out of 14 sera were P.T. positive, five out of seven in the 30-39 age-group, and all sera from persons over the age of 40 were positive. There were five specimens from persons who had been jaundiced for at least 10 days which gave negative mouse protection tests, thus excluding the diagnosis of yellow fever in those persons, and the P.T. positive sera were distributed almost equally between jaundiced and apparently healthy individuals. It thus appears that the jaundice was not due to yellow fever. Comparison of the H.A.I. and P.T. results shows 14 disagreements out of 41 sera, 11 of these being H.A.I. positive but P.T. negative.
Area V. Tamale. Tamale, the administrative centre of the Northern Territories, is a medium-sized market town situated on a low hill in the centre of country ranging from guinea savannah to true savannah. The last known outbreak of yellow fever was in 1931 when six Europeans died, the infection being apparently introduced from Pong Tamale 20 miles further north. The villages of Chogu and Yohni were visited and 96 sera were collected. These were examined in separate protection tests in which the levels of yellow fever virus employed were respectively 562 and 158 LDS0. The results in the two villages were very similar, and have been combined in the analysis. The youngest persons having positive sera were two females aged 25 in Chogu village, out of a total of 15 examined in that age-group. Three out of 14 sera were positive by P.T. in the 30-39 age-group, and 14 out of 15 in the group aged 40 and above. These findings are in agreement with the known history of yellow fever and suggest that there have been no further introductions of the disease since 1931. Two sera from children gave positive H.A.I. tests ; one was from a femal e aged 2 whose serum gave titres of 10 and 12.5 in two tests ; the other was from a boy of 14 which gave titres of 12.5 and 20. Otherwise the H.A.I. results are in close agreement with those obtained by P.T. Detailed results are presented in Fig. 3. Area VI. Pong Tamale. Thirty-nine specimens were collected in the native village of Pong Tamale. Apart from one serum from a'girl of 10, all specimens from persons under the age of 27 gave negative protection test results, whilst of sera from 16 persons over that age only one was negative and one doubtful. All 16 specimens from persons over 27 were also H.A.I. positive, as was that from the 10-year-old girl. The 11 specimens which were H.A.I. positive, but P.T. negative, were mostly from children, the youngest being aged 3½. Area VII. Bolgatanga. The two villages of Bongo and Yorogo, less than 10 miles south of the northern boundary of the Gold Coast, were selected as more suitable for survey purposes than the town of Bolgatanga itself. Both villages are in open, rocky country, with wide areas of grassland and only occasional stunted trees or bushes. Enquiry produced no history of yellow fever in Bolgatanga ; the nearest recorded cases of yellow fever were in a Syrian who died at Wale
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FIG. 4. A r e a V I I . Bolgatanga. I n each of F i g u r e s 2, 3 a n d 4, t h e lower section shows t h e results of b o t h P . T . a n d H . A . I . tests o n i n d i v i d u a l sera a r r a n g e d a c c o r d i n g to age-groups. I n each a g e - g r o u p t h e left c o l u m n r e p r e s e n t s P . T . results a n d t h e r i g h t c o l u m n H . A . I . results. A n o p e n s q u a r e indicates a negative result ; a closed square, a positive result ; a n d a h a t c h e d square, a d o u b t f u l result. T h e central section shows t h e p e r c e n t a g e of positive tests in each age-group. Solid l i n e - H . A . I . test, d o t t e d l i n e - - P . T . T h e u p p e r section shows t h e g e o m e t r i c m e a n s of t h e titres of sera giving positive H . A . I . tests.
352
I-IAEMAGGLUTINATIONTEST IN YELLOWFEVER
Wale, 35 miles south of Bolgatanga, in 1931, and two Europeans who died in Lawra and T u m u in 1936 and 1938. These two towns are on the same latitude as Bolgatanga but 140 and 80 miles west, respectively. Sixty-four sera were collected in all, and the results from the two villages have been combined. T h e youngest subjects from whom definitely positive sera were obtained were four young men of 25 ; above that age, sera from all but three out of 26 persons gave positive protection tests. An interesting feature of the survey in this area was the high proportion of doubtful results, not less than 11 out of 64 in the original tests. T h e doubtfuls are not due to the low dose of virus in one test (43 LD50), since they occur about equally in the two villages in separate runs of protection tests. W h e n nine of these were re-tested, one gave a clear positive result, five gave negative results and three remained doubtful in a test employing 186 L D 5 0 of yellow fever virus. Fig. 4 gives detailed results of H.A.I. and P.T. results and indicates the age distribution of the specimens. It will be seen that H.A.I. positive sera occur in the youngest age-group tested and increase in frequency with age, so that only one specimen from a person over the age of 20 is H.A.I. negative. HAEMAGGLUTINATION I N H I B I T I O N TITRES
T h e results so far presented have been based upon a simple positive or negative reading of the H.A.I. test, depending upon the presence or absence respectively of inhibitory substance in a 1 : 10 dilution of serum extract. In practice all sera were first tested in dilutions of up to 1 : 40 since the presence o f a prozone phenomenon indicated that a screening test on sera at 1 : 10 dilution only might miss a proportion of positives. Subsequently all positive extracts were re-tested in further dilutions to reach an endpoint. Such tests were repeated a second time and the geometric mean of the two titres was taken as the final result. T h e reproducibility of the test was analyzed by tabulating the differences between the two observations. Of 125 pairs of observations, the mean difference was 0.03, and the standard deviation 0.90, where 1.0 equals one twofold dilution. Since the haemagglutinating dose varied from 10 to 20 in different tests, and the titre of a serum bears a linear relationship to the test dose, the reproducibility of the result is reasonably good. T h e geometric means of the H.A.I. titres of sera from the different areas are included in T a b l e I. In Areas I, II, III, IV, and V the titres are all generally low and show no particular pattern. In Areas VI and VII, in the Northern Territories, the sera show a steady increase in H.A.I. titres with advancing age. DISCUSSION
In Areas I, II, I I I and V, the two tests show an agreement which is statistically highly significant. Taking the results Of these four areas together, the over-all agreement between the two tests is 91 per cent. of 230 sera examined. In these areas then, the yellow fever H.A.I. test appears to give a fairly reliable indication of the immunity state of the" population as determined by the mouse protection test, and cross immunity due to the presence of other viruses does not complicate the interpretation of the results. In Area IV, Kpandae, the agreement between the two tests is less significant. At first it appeared possible that the rather high level of 900 L D 5 0 of virus used in the protection test might have explained some of the H.A.I. positive, but P.T. negative, findings. However, when certain of these were re-tested with a lower L D 5 0 they remained negative. T h e
J. s. PORTERFIELD
353
Kpandae sera are not, however, a random selection since they include specimens from persons bled in convalescence from an undiagnosed illness. It is possible that the agent responsible for the jaundice may have caused the excess of H.A.I. positive sera found in this group ; the evidence on this point is not conclusive. Whatever the explanation, it is clear that with this particular set of sera, collected at this particular time, the H.A.I. test is an unreliable measure of yellow fever immunity as determined by mouse protection tests. At Pong Tamale the agreement is poor, and at Bolgatanga only 53 per cent. of all the sera give the same result by both tests. It has already been noted that these two areas differ from the rest in that they show increasing H.A.I. titres with advancing age. The absolute levels of H.A.I. titres in these areas are higher than would be expected if there had been no yellow fever for about 20 years, as the P.T. results seem to suggest. The most reasonable explanation for these two observations would seem to be the presence in the north of the Gold Coast of an agent or agents serologically related to yellow fever virus which produces antibody cross reacting with yellow fever antigen in H.A.I. tests. So little is known of the distribution of the various tropical viruses throughout West Africa, that it is difficult without testing sera against all the known possible agents, to determine which might be responsible. Further studies on this problem are proceeding. SUMMARY
(1) Throughout the southern regions of the Gold Coast, agreement between yellow fever P.T. and H.A.I. results is sufficiently close for the latter to be used as an indication of immunity levels in the population. Near the northern boundaries of the Gold Coast this relationship does not hold, and reliance on H.A.I. results would give very misleading results. (2) To guard against possible confusion due to the presence of serologically related viruses, it is suggested that every fifth serum from adults, and possibly all sera from children, showing a positive H.A.I. result, should be checked by yellow fever protection tests. Similar or more frequent checks should be carried out in areas where there is evidence of recent virus infection. REFERENCES CASALS, J. & BROWN,L. V. (1954). J. exp. Med., 99, 429. DENTON, J. E. • BELCHER,H. K. (1949). J. Amer. reed. Ass., 141, 1051. PORTERFIELD, J. S. (1954). Trans. R. Soc. trop. Med. Hyg., 48, 261. (1955). Proceedings VIIth International Congress of Comparative Pathology. Lausanne, 1955. SAWYER, W. A. ~x;LLOYD,W. (1931). J. exp. Med., 54, 533.
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