Measurement of malarial infectivity of human ...

Parasitology (1988), 96, 251-263 With 2figuresin the text

251

Measurement of malarial infectivity of human populations to mosquitoes in the Madang area, Papua New Guinea P. M. GRAVES 1 *, T. R. BURKOT 1 , R. CARTER2f, J. A. CATTANPJ, M. LAGOG1, J. PARKER 1 , B. J. BRABIN 1 , F. D. GIBSON 1 , D. J. BRADLEY 3 and M. P. ALPERS 1 1

Papua New Guinea Institute of Medical Research, P.O. Box 378, Madang, Papua New Guinea 2 Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md 20792, USA 3 London School of Hygiene and Tropical Medicine, Keppel St, London WCIE 1HT, England (Accepted 1 October 1987) SUMMARY The proportion of blood meals taken on humans which are infectious to mosquitoes in the Madang area, Papua New Guinea was estimated by two methods. In the first, laboratory reared Anopheles farauti were fed on individuals of all ages at village surveys. The results showed that 3 8 % of people were infectious and that the mean percentage of mosquitoes which became infected by feeding on these people was 37-9%. From the average proportion of mosquitoes infected, the probability that a mosquito feeding on a human would pick up infection was 0-013 + 0-005. In the second approach mosquitoes were fed on identified Plasmodium falciparum, P. vivax and P. malariae gametocyte carriers. The results indicated that 46 % of gametocyte carriers were infectious and that the mean probability of a mosquito becoming infected after feeding on a gametocyte carrier was 0-151+0-029. Gametocyte prevalence rates in all ages measured over 18 months in three villages averaged 3 3 % P. falciparum, 4-0% P. vivax and 0-7% P. malariae, totalling 8-0 + 0-7%. Combining gametocyte prevalence rates with the probability of a mosquito becoming infected from a gametocyte carrier, the probability of a mosquito becoming infected following a blood meal on a member of the human population was estimated to be 0012 + 0003.

INTRODUCTION

The object of this investigation was to study the malarial infectiousness of human populations to mosquitoes in the Madang region of Papua New Guinea. Infectiousness of a human population can be thought of as the proportion of the human population which at any one time is capable of infecting mosquitoes. Alternatively it can be defined as the proportion of mosquitoes biting a human population on any one night which acquire an infection. In the present paper we use the second meaning for infectiousness and define it as the mosquito infection probability (K). A measure of the mosquito infection probability is of both theoretical and practical interest for understanding the epidemiology of malaria. The impact of a malaria vaccine * Present address: Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane, Queensland, Australia 4006. f Present address: Institute of Animal Genetics, West Mains Rd, Edinburgh EH9 3JN, Scotland. j Present address: Department of Tropical Public Health, Harvard School of Public Health, 665 Huntingdon Ave., Boston, MA 02115, USA.

252

P. M. GRAVES AND OTHERS

on the infectiousness of a human population to mosquitoes (K) will determine its ultimate effect on the transmission of malaria in that population. Development of a practical method for measuring K could facilitate monitoring the effectiveness of malaria intervention strategies, including vaccines. Various ways of measuring K are feasible. A direct approach is to feed groups of mosquitoes on a demographically representative human population (without regard to parasitological status). A value will then be obtained for the proportion of individuals capable of infecting mosquitoes and, among such infectious individuals, of the proportion of mosquitoes which become infected. From these data a value for K can be estimated. Another approach is to determine the prevalence of gametocyte carriers in a human population by blood-slide surveys, while data on infectivity of gametocyte carriers are independently obtained by feeding mosquitoes on known gametocyte carriers. The infection probability K is then derived by multiplying the mean probability of infection (for the gametocyte carriers tested) by the prevalence of gametocyte carriers in the human population. Results from these two approaches for villages near Madang, Papua New Guinea are presented in this paper. The villages lie within the study area of the Papua New Guinea Institute of Medical Research, which has conducted a multidisciplinary study of malaria in the area (Cattani, Tulloch, Vrbova, Jolley, Gibson, Moir, Heywood, Alpers, Stevenson & Clancy, 19866). Entomological and parasitological data from particular villages have been described (Cattani, Moir, Gibson, Ginny, Paino, Davidson & Alpers, 1986a; Burkot, Graves, Cattani, Wirtz & Gibson, 1987; Charlwood, Graves & Birley, 1986). Because of marked variation in prevalence within the area (Cattani etal. 19866; Burkot et al. 1987), attention has been paid to examining variation in gametocyte rates between villages. MATERIALS AND METHODS

In the villages of Butelgut, Mebat and Maraga, six parasitological surveys were carried out between June 1983 and September 1985. Four surveys at 3-monthly intervals were followed by two surveys at 9-monthly intervals. Blood slide surveys were also conducted in the villages of Sah, Umun, Bahor and Buksak at the same time as mosquito infectivity studies. Survey procedures were as described by Cattani et al. (19866), except that after the initial reading of the slides to determine prevalence of P. falciparum, P. vivax and P. malariae asexual stages and P. falciparum gametocytes, they were later re-examined for P. vivax and P. malariae gametocytes. During surveys in Mebat, Umun and Bahor in 1984, infectivity of individuals to mosquitoes was studied by membrane feeding. The laboratory reared mosquitoes used were from a colony of Anopheles farauti established from a cross between wild-caught females from the village of Agan, Madang province, and males of a colony of An. farauti No. 1 from Rabaul, East New Britain province, provided by Dr Tony Sweeney. At the village surveys fingerprick blood samples of at least 200 /A were drawn into heparinized containers and the blood immediately presented to starved An. farauti through waterjacketed (37 °C) glass feeders using Baudruche membrane. Because of the reluctance of An. farauti to feed through membranes, in 1985 villagers in Butelgut, Mebat, Sah and Buksak were asked to allow groups of 20-30 mosquitoes to feed directly on themselves. Membrane or direct feeds were also performed on gametocyte carriers identified amongst outpatients at Yagaum and Alexishaven Health Centres, Madang Province,

Malarial infectivity in Papua New Guinea

253

Table 1. Mosquito feeds at village surveys in Madang Slide result Gametocytes P. falciparum P. vivax P. malariae Asexuals only P. falciparum P. vivax P. falciparum + P. vivax P. malariae P. falciparum + P. ovale No parasites any stage Total Percentage infectious

0-4 years

Number of feeds performed (no. infectious) > = 20 years 5-9 years 10-19 ye 2 1 2(2)

3(1) 2(1) 1

11 2(1) 2 0 1

18 6(1) 2 0 0

16 5 0 0 0

10

14

30

19(1)

35 (3)

62 (3)

53

8-6

4-8

63 85 (1) 1-2

1 3(1) 1 3 0 1 0 0

0 1 (1) 0

Total 6(1) 7 (3) 4(2) 48 13 (2) 5 0 1 117 201 (8) 40

and at Goroka Hospital, Eastern Highlands Province. Since malaria transmission does not occur in Goroka, gametocyte carriers had acquired their infections in a coastal area. In all cases, blood-slides were taken at the time of feeding and subsequently examined for parasite species and density determination. Immediately after being offered blood, mosquitoes were sorted and unfeds were discarded. Engorged mosquitoes were held in an insectary at ambient temperature (mean 27-28 °C) for 7-9 days, with a continuous supply of 10% sucrose solution. Their stomachs were then dissected, stained with 0 - 2% mercurochrome and examined at x 400 for oocysts. On occasions when some mosquitoes in a group were left for more than 9 days, oocyst infections developed normally in the mosquitoes of this colony and sporozoites were produced. RESULTS

Mosquito feeds on the village populations Groups of mosquitoes were dissected from feeds performed on 201 individuals (Table 1), of which 8 (4>0%) gave rise to infections in mosquitoes. The mean percentage of mosquitoes infected in feeds on these 8 infectious individuals was 37-9% (range 9-l—75-0%). The average number of mosquitoes dissected/feed was 8*0 (s.D. = 3-5). It is possible to estimate the mosquito infection probability, K, by multiplying the above two proportions (0379 x004) giving 0015. However, because of the standard error arising from such a multiplication, it was preferable to estimate K from the average of the proportion infected in each group (weighted by sample size) as 0*013+ 0-005. As a third alternative, dividing the total number of mosquitoes infected (21) by the total dissected (1610) gave an estimate of 0-013 + 0-003 for the mosquito infection probability. On subsequent examination of the blood slides, 17 persons (8-5%) were observed to have gametocytes (any species), and 6 of these people were infectious. However, out of the 12 persons in whose slides only P. vivax asexual stages were reported, 2 were infectious to mosquitoes, indicating that in these cases P. vivax gametocytes had been undetected. In no cases were infected mosquitoes obtained from persons whose slides were reported to show P. falciparum, P. malariae or P. ovale asexual stages only, or no

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Table 2. Demographic profile of study villages compared to mosquito feeds on each age group

No. of people No. of mosquito feeds

Age group (years) 10-19

0-t

5-9

89(16-3%) 19 (9-5%)

79(14-5%) 35(17-4%)

129(23-7%) 62(30-8%)

> = 20

Total

248(45-5%) 85(42-3%)

545 201

Table 3. Average parasite prevalence by age in Butelgut, 1983-5 Species P. falciparum Asexuals Gametoeytes P. vivaz Asexuals Gametoeytes P. malariae Asexuals Gametoeytes P. male. Overall positive N

0-4

Age group (years) 5-9 10-19 > = 20

Total

41-8 90

50-6 6-7

46-9 3-4

24-3 20

36-6 40

10-4 90

22-5 12-5

7-6 21

4-8 00

9-1 36

00 0-0 0-0 493 67

11 0-0 1-1 65-2 89

21 0-7 0-7 510 145

00 00 00 29-1 251

0-7 0-2 0-4 43-1 552

parasites of any stage. Only 1 of the 54 P. falciparum cases (any stage) was infectious whereas 5 of the 20 P. vivax cases were infectious. The distribution of individuals in the different age categories upon whom feeds were successfully peformed was compared with the demographic profile of the population at large. Accurate, up-to-date demographic data from house-to-house surveys during 1985 was available for the villages of Butelgut, Mebat, Maraga and Sah (Table 2). There was a significant difference between the demographic profile of the population on which mosquitoes fed and the demographic profile of the population as a whole (heterogeneity X2 = 8*91, P = 0'031). This arose from a significant under-representation of 0 to 4-yearolds in feeding studies compared to their numbers in the population (x2 = 5*07, P = 0-024), due to the refusal of many young children to allow mosquitoes to feed on them. After correcting for the distribution of feeds on different age groups, the proportion of persons infectious was estimated as 3'8%. Parasitological results Plasmodium faciparum, P. vivax, P. malariae and P. ovale prevalences by age group in the villages of Butelgut, Mebat and Maraga in 1983-5 are presented in Tables 3-5. There was no significant difference between the three villages in the crude prevalence of P . falciparum asexual stages. However, the prevalence in the 0—4 age group was significantly lower in Maraga than in Mebat (x2 = 511, P = 0-02). In the 10-19 years age group, the prevalence in Mebat was significantly lower than in Butelgut (x2 = 59, P = 0015) and close to significance compared to Maraga (x2 = 3-56, P — 0059). Gametocyte rates of P. falciparum ranged from 2-2 % in Maraga to 4-0 % in Butelgut.

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Table 4. Average parasite prevalence by age in Mebat, 1983-5 Age group (years) > = 20 10-19

Tota

Species

0-4

5-9

P. falciparum Asexuals Gametocytes

45-6 7-8

520 50

30-8 1-9

22-5 20

34-3 3-6

22-2 12-2

260 130

10-6 1-9

8-3 10

14-9 5-6

11 00 10 56-7 90

100 51 00 690 100

4-8 1-9 00 42-3 104

2-5 00 00 33-3 204

4-2 1-4 0-2 46-6 498

P. vivax Asexuals Gametocytes P. malariae Asexuals Gametocytes P. ovale Overall positive N

Table 5. Average parasite prevalence by age in Maraga, 1983-5 Species P. falciparum Asexuals Gametocytes P. vivax Asexuals Gametocytes P. malariae Asexuals Gametocytes P. ovale Overall positive N

Age group (years) > = 20 10-19

Total

0-4

5-9

28-8 5-8

42-6 11

44-3 1-9

17-6 11

30-5 2-2

8-7 2-9

25-5 9-6

113 10

11 0-5

9-6 2-9

10 10 00 33-7

3-2 11 0-0 62-8 94

0-9 00 00 53-8 106

0-5 00 00 19-3 187

1-2 0-4 00 381 491

104

There was no significant difference between the villages in the P.falciparum gametocyte rate, either overall or in any of the age groups. The overall prevalence of P. vivax infections was significantly higher in Mebat than in Butelgut (#2 = 791, P = 0-005) and Maraga (x* = 595, P = 0015). The higher rates in Mebat were most marked in the 0-4 and the over 20 age groups. However, P. vivax gametocyte rates were not significantly different between villages. Rates of P. malariae infection were also higher in Mebat than in Butelgut (x2 = 1228, P = 00005) and Maraga (#2 = 7-26, P = 0007). The prevalence of gametocytes (all species) was 7-3 % in Maraga, 7-8 % in Butelgut and 10-4% in Mebat. These differences are not significant. The overall prevalence of gametocytes in the three village populations was 3-3% P. falciparum, 4-0% P. vivax and 0-7% P. malariae, giving a combined gametocyte prevalence of 8 + 0-7%. The gametocyte rates of P. falciparum and P. vivax were not significantly different. The peak prevalence of P. falciparum was observed in the 5—9 year age group in Butelgut and Mebat but in the 10-19 year age group in Maraga. Prevalence rates of P. vivax peaked in the 5-9 year age group in all three villages. For P. falciparum, gametocyte rates were always highest in the 0-4 age group.

256

P. M. GRAVES AND OTHERS P. falciparum

P. vivax

P. malariae

50 -,

40

.1 o

30

10

— ~J 0-4

5-9

10-19

>20

0-4

5-9

10-19

>20

0-4

5-9

10-19

>20

Age (years)

Fig. 1. Prevalence of asexual stages ( ) and gametocytes ( ) of Plasmodium falciparum, P. vivax and P. malariae by age group in the villages of Butelgut, Mebat and Maraga combined, 1983-5. Bars represent binomial standard errors.

The overall prevalences for all three villages combined are shown in Fig. 1. The P. falciparum prevalence was 3 times higher than P. vivax and 16 times greater than P. malariae; P. ovale represented less than 1 % of all infections. Relative ratios between infections with sexual and asexual stages were obtained from the prevalences for each age group shown in Fig. 1. Despite highest asexual prevalence in 5 to 9-year-olds, the relative ratio between P. falciparum infections with gametocytes and those with asexuals only declined sharply from 193% in 0 to 4-year-olds to 8 7 % in 5 to 9-year-olds. In P. vivax the gametocyte rate was higher in the 5-9 year age group than in 0 to 4-year-olds, but the relative proportion of P. vivax infections which had gametocytes was highest in the 0-4 age group (55-8%) and declined through the subsequent age groups to 10-4% in the over 20 age group. Examination of 1541 blood-slides taken in Butelgut, Mebat and Maraga during 1983—5 revealed a total of 51 infections with P. falciparum gametocytes, 62 with P. vivax gametocytes, and 10 with P. malariae gametocytes. These numbers are insufficient to investigate the distribution of parasite densities by age groups. Therefore P. falciparum gametocyte and asexual densities by age were analysed using a larger sample of blood-slide surveys undertaken in Madang during 1983-5, in which there were a total of 158 gametocyte and 1133 asexual infections (Table 6). Despite highest prevalence rates of P. falciparum asexual stages in 5 to 9-year-olds (Fig. 1), average asexual parasite density grade showed a decline with age from 3-7 in 0 to 4-year-olds to 1*8 in over 20-year-olds. There was a corresponding decline in gametocyte density grade through the age groups (Table 6). The decline in P. falciparum density with age is clear in Fig. 2; in the case of P. falciparum gametocytes the majority of infections were in the lowest density grade. However, the proportion of infections with density greater than grade 2 was significantly higher in the 0 to 4-year-olds than in the other age groups (X2 = 914, P = 0003).


257

Table 6. Average Plasmodium falciparum density grade by age, 1983-5

P. falciparum Asexuals Mean density S.D.

N P. falciparum. Gametocytes Mean density S.D.

N

Age group (years) 10-19 > = 20

0-4

5-9

Total

3-65 2-30 (211)

3-42 2-18 (289)

2-43 1-83 (343)

1-83 1-47 (290)

2-76 2-07 (1133)

1-46 0-87 (56)

116 0-75 (43)

1-20 0-66 (30)

107 0-37 (29)

1-26 074 (158)

Parasite density grades: (parasites/mm 3 ) 0 = negative, 1 = < 80, 2 = 81-200, 3 = 201-600, 4 601-1400, 5 = 1401-3400, 6 = 3401-8000, 7 = 8001-19960, 8 = > 19961.

5-9

D-4

1 0 -|

> 20

10-19

years

A 0-80-60-4= 0-2-

s

"1

•

o c o

a o

B

i-0-i 0-8 • — 0-60-40-2 •

n,

1 2

3 4

1 2

3 4

12

3 4

1 2

3 4

Density grade

Fig. 2. Frequency distribution of densities of asexual stages (A) and gametocytes (B) in Plasmodium falciparum infections by age group in the Madang study area, 1983-5. The number in each sample of asexual and gametocyte infections respectively was as follows: 0-4 years: 211, 56; 5-9 years: 289, 43; 10-19 years: 343, 30; Js 20 years: 290, 29. The density grades were as follows (parasites/mm 3 ): 1 = 0-200, 2 = 201-1400, 3 = 1401-8000, 4 = > 8000.

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Table 7. Results of mosquito feeds on Plasmodium falciparum gametocyte carriers Parasite density { Placef

Type of feed

G M* M M M M M M* M G M* M* M M* M* G M M M G G M M M G M G M M G M

Membrane Direct Membrane Membrane Membrane Membrane Membrane Direct Membrane Membrane Membrane Membrane Membrane Membrane Direct Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Membrane Direct Membrane

Age (years) 50 10 8 2 4 3 9 16 5 25 2 8 4 7 15 35 ?

8 3 25 39 30 0 ?

17 2 16 3 % 50 7

Pfa

Pfg

No. of mosquitoes dissected

0 0 0 0 0 0 0 0 1 1 1 1 1 5 5 6 0 0 0 4 8 0 0 1 1 1 6 8 0 7 8

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 4 4 4 4 4 4 4 5 5 5

33 4 18 6 7 8 7 13 4 11 7 10 7 5 13 18 7 10 4 12 10 20 5 5 37 8 13 6 14 8 7

No. infected 0 0 1 0 1 0 0 0 0 7 0 0 1 0 3 2 1 0 1 0 0 0 0 0 2 1 13 2 4 0 1

Mean oocysts/ infected mosquito — — 20 — 10 — — — — 30 — — 10 — 1-3 3-5 10 — 10 — — — — — 10 10 54-2 5-5 6-5 — 10

* Feed at village survey, included in Table 1. f Location: M = Madang; G = Goroka. % Parasite density: 0 = negative, 1 = < 80, 2 = 81-200, 3 = 201-600, 4 = 601-1400, 5 = 14013400, 6 = 3401-8000, 7 = 8001-19960, 8 = > 19961 parasites/mm3. Pfa = P. falciparum asexual stages; Pfg = P. falciparum gametocytes.

Mosquito feeding on known gametocyte carriers Feeds on 31 P. falciparum gametocyte carriers revealed that 45*2% of them were infectious (Table 7). The average number of mosquitoes dissected/group was 10-9. The overall proportion of mosquitoes infected in the total of 337 fed was 0119 + 0018. Since there was extreme heterogeneity in the degree of infectiousness of different people, the mean proportion of mosquitoes infected was calculated by averaging the proportion infected in each group, giving a value of 0118 + 0039. The mean number of oocysts/ infected mosquito ranged from 1-0 to 54-2 in the different groups. For P. vivax (Table 8), 14 out of 28 feeds were infectious (50-0%). These include 3 feeds on P. vivax-infected people in whose slides gametocytes were undetected and 3 feeds on persons whose slides were not examined for gametocytes. The overall


259

Table 8. Results of mosquito feeds on Plasmodium vivax gametocyte carriers Parasite density J

Placet

Type of feed

M* M M* M* M* M* M* M* M* G G M* M G G M G G G G G G M G G M G G

Direct Membrane Direct Direct Direct Membrane Direct Direct Membrane Direct Membrane Membrane Direct Direct Membrane Membrane Direct Membrane Membrane Membrane Direct Membrane Membrane Membrane Membrane Direct Membrane Membrane

Age (years) 18 1 9 17 54 4 4 10 8 16 16 3 12 27 17 5 50 30 20 14 18 ? 2 35 22 7 20 25

No. of mosquitoes Pfa Pva Pvg dissected 0 0 8 0 0 5 1 3 4 5 6 0 0 0 0 3 0 0 0 0 3 6 0 0 8 3 0 0

1 8 3 1 1 1 3 3 3 4 4 5 5 5 5 5 6 6 6 6 7 7 8 8 4 4 6 7

() () ()

1 5j 5>

10 24 6 9 4 11 4 6 6 4 16 11 4 9 10 16 3 9 16 17 5 16 52 19 13 8 23 15

No. infected

Mean oocysts/ infected mosqui

3 2 2 4 3 0 2 0 0 0 0 0 3 0 0 0 2 1 2 0 0 0 2 0 0 4 14 7

1-3 8-3 20 1-8 1-3 — 10 — — — — — 40 — — — 31-5 10 10 — — — 3-8 — — 3-3 50 41

* Feed at village survey, included in Table 1. f Location: M = Madang, G = Goroka. j Parasite density: 0 = negative, 1 = < 80, 2 = 81-200, 3 = 201-600, 4 = 601-1400, 5 = 14013400, 6 = 3401-8000, 7 = 8001-19960, 8 = > 19961 parasites/mm3. Pfa = P. falciparum asexual stages, Pva = P. vivax asexual stages, Pvg = P. vivax gametocytes.

proportion of mosquitoes infected was 0-147+0-019, but again to allow for heterogeneity between individuals the mean proportion infected was obtained by averaging the probabilities in each group, giving an estimate of 0-203 + 0-051. The average number of mosquitoes dissected was 12-4. The mean number of oocysts/infected mosquito ranged from 1-0 to 31-5. The presence of a P. falciparum asexual infection in conjunction with P. vivax appeared to reduce the infectivity of P. vivax cases: only 3 out of 12 such mixed infections were infectious compared to 11 out of 16 of the cases with P. vivax only (X2 = 3-65, P = 0056). Table 9 shows data from all feeds performed on P. malariae-infected cases. In all except 2 of them, gametocytes were detected. Four out of 11 gametocyte carriers (36-4%) produced infections in the mosquitoes; the overall proportion of mosquitoes infected was 0072±0026. The mean proportion infected/group was 0111 ±0062. The mean number of mosquitoes fed was 106 and the mean number of oocysts/infected mosquito ranged from 1-0 to 7-0.

260


Table 9. Results of mosquito feeds on Plasmodium malariae infected individuals Parasite density}:

Age

No. of mosquitoes Pfa PmaPmg dissected

No. Mean oocysts/ Type of feed (years) infected infected mosquito — M Membrane 4 0 0 30 3 0 4 M 2 10 0 Direct 3 3 3 — 1 1 M 8 4 0 0 Direct 1 1 11 M* 10 3 9 0 Direct 1 14 Direct 4 7 M* 3 7-0 0 — 1 M Membrane 3 0 5 6 0 — 1 M Membrane 11 7 0 5 0 4 2 4 M Direct 1 10 8 0 — 2 8 M Membrane 6 8 0 0 — 2 1 1 7 10 M Direct 0 — 1 4 12 M* 3 18 Direct 0 4 4 11 2 M* 0 0 Direct * Feed at village survey, included in Table 1. t Location : M = Madang, G = Goroka. % Parasite density: 0 = negative, 1 = < 80, 2 = 81-200, 3 = 201-600, 4 = 601-1400, 5 = 14013400, 6 = 3401-8000, 7= 8001-19960, 8 = > 19961 parasites/mm3. Pfa = P. falciparum asexual stages, Pma = P. malariae asexual stages, Pmg = P. malariae gametocytes.

Placet

The influence of parasite density and age on infectiousness was investigated by logistic regression using the GLIM statistical package. In the case of P. falciparum, there was a statistically significant difference between infectiousness in the different classes of gametocyte density (^3 = 9-3, P < 0-05), but there was no trend of infectiousness increasing or decreasing with parasite density. However, if the one case in which 100% of mosquitoes were infected was excluded, the effect was no longer significant (#§ = 3-85). Analysis of the effect of age on infectiousness, which was restricted to cases in the endemic area of Madang (23 cases), showed no significant effect (xl = 1'36). Similar analysis of the effect of gametocyte density on P. vivax infectiousness was not possible because of the restricted number of gametocyte density classes. In the Madang feeds (14 cases), infectiousness of P. vivax increased with age (^3 = 14-84), although the limited data available means that this result should be interpreted with caution. Similar lack of sufficient data precluded analysis of factors affecting P. malariae infectiousness. There was no significant difference between the three malaria species in the proportion of gametocyte carriers who were infectious to mosquitoes, which averaged 45-7 %. Nor was there a significant difference in the proportion of mosquitoes infected/ group by the three malaria species. An overall estimate of mean proportion infected, obtained by averaging the proportion infected/group, was 0-151+0-029. Multiplying this proportion by the prevalence of gametocyte carriers in the population, which was 0-080 + 0-007 (see above), gave an estimate for mosquito infection probability of 0012 ±0003. Of the P. falciparum gametocyte carriers from Madang (Table 7), the numbers in each age group, and the percentage infectious, were as follows: 0-4 years, 9 (56%); 5-9 years, 7 (29 %); 10-19 years, 3 (33 % ) ; ^ 20 years, 1 (0 %). For P. vivax, the figures were as follows: 0-4 years, 5 (60%); 5-9 years, 4 (50%); 10-19 years, 4 (75 % ) ; ^ 20 years, 1 (100%).

Malarial inactivity in Papua New Guinea

261

The frequency distributions of oocyst numbers in infected mosquitoes were markedly non-normal, even after logarithmic transformation and, in calculating the overall means, counts of over 20 oocysts (9 cases) were excluded. Geometric mean oocyst number/infected mosquito for P. falciparum was 350 (N = 32) and for P . vivax was 349 (N = 52, t = 0-021,N.S.). Median values were 3 oocysts for P. falciparum and 2 oocysts for P. vivax.

DISCUSSION

Feeding of laboratory reared mosquitoes on people of all ages in the population was performed in an attempt to determine the proportion of persons in a malaria endemic area who are infectious to mosquitoes. The only previous study of this kind was performed by Muirhead-Thomson (1957) in West Africa, in which he found that 10-11% of individuals were infectious. After correcting for the demographic constitution of the population on whom feeds were performed, 3-8% of the population were estimated to be infectious in our study. However, not all mosquitoes feeding on an ' infectious' person become infected; and the probability of a mosquito becoming infected after feeding on a person (K) was estimated as 0013 + 0-005. Such studies involving feeding mosquitoes on large numbers of people are hampered by logistical problems, including the reluctance of laboratory-reared mosquitoes to feed and reluctance of the population, particularly children, to allow mosquitoes to feed on them. A second approach to estimating infectivity was therefore adopted which involved feeding of mosquitoes on gametocyte carriers to measure their infectivity. The results obtained were combined with data on the prevalence of gametocyte carriers in the population to estimate the infectivity of the population at large. It was observed that 0-151+0-029 of the mosquitoes feeding on a gametocyte carrier (any species) would become infected. Since the gametocyte rate (all species) was 0-080 + 0-007, the mosquito infection probability, K, was estimated as the product of these two proportions: 0012±0-003. Thus both methods gave similar estimates of 0-012-0-013 for the proportion of mosquito feeds on humans which pick up infection. The fact that certain individuals with high gametocyte densities did not infect mosquitoes indicated that gametocytes are impaired or otherwise prevented from infecting mosquitoes in these individuals. It is unlikely that this is due to the sporonticidal effects of antimalarial drugs since Fansidar was not available to these study groups through local health services. Data from feeding P . falciparum gametocytes from in vitro cultures to mosquitoes by membrane feeding have shown that many sera from individuals in Papua New Guinea suppress infectivity of parasites to mosquitoes (Graves, Carter, Burkot, Quakyi & Kumar, 1987). A strong negative correlation was shown between the infectivity of these sera and the presence of antibodies against P. falciparum gamete surface antigens. The presence of such antibodies was not age related and could well account for the failure of many gametocyte carriers in any age group to infect mosquitoes. There are similar findings of transmission-blocking antibodies in P. vivax infections (Mendis, Munesinghe, de Silva, Keragalla & Carter, 1987). Gametocyte rates observed in the population around Madang are much lower than is commonly reported for Africa (Muirhead-Thomson, 1954; Molineaux & Gramiccia, 1980). More pertinently, rates are much lower than were observed in the East Sepik Province, Papua New Guinea during the 1950s by Peters (1957) who reported a gametocyte rate (all species) of 4 1 5 % in under 2-year-olds, which declined with age but

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remained at 12-8% in adults. The highest rate observed in the present study was 18% in 5 to 9-year-olds, and the overall rate was 8%. In a comparable area of Irian Jaya near the Papua New Guinea border, Metselaar (1957) found the P. falciparum gametocyte rate to be 306% in 2-year-olds, declining to 94% in 6 to 8-year-olds and 1-3% in the over 14-year-olds. One reason for the lower gametocyte rate currently observed in 0 to 4-year-olds around Madang could be the easy availability of chemotherapy in the area (Cattani et al. 19866; Moir, Tulloch, Vrbova, Jolley, Heywood & Alpers, 1986), which could result in faster elimination of parasites and/or lower density infections. In the present study only approximately 10% of P. falciparum infections went on to produce gametocytes, whereas Peters (1957) stated that approximately 30% of infections produced gametocytes. The parasitological data presented here demonstrate that gametocyte prevalence declines with age as immunity to the asexual parasites increases. The data further indicate that gametocytes of P. falciparum and P. vivax decline in prevalence relative to asexual stages, even when the latter is rising, as in the 5-9 year age group. This suggests that some form of acquired immunity may be directly affecting the production of gametocytes and/or their survival. In P. falciparum the proportion of gametocyte carriers who were infectious was higher in the 0 to 4-year-olds than in the over 5-yearolds, as would be expected given the difference in gametocyte density. It is interesting to compare the proportion of infections found to contain gametocytes in this study with the rates observed by Metselaar (1960) during a spraying campaign in Irian Jaya. After spraying, there was a marked increase in the proportion of gametocyte-positive infections. Thus, in 3 to 8-year-olds, the proportion of infections with gametocytes increased over a 5-year period from 7 % to 40 % for P.falciparum and from 9% to 50% for P. vivax. This increase, which in general was most marked in P'. falciparum, gave rise to a shift in the proportion of infections accounted for by this species. This change was attributed to a loss of immunity by the population and subsequent increased proportion of infections which gave rise to gametocytes. This emphasizes the importance of considering the infectiousness of the population in malaria control projects or vaccination campaigns, since the effect of an intervention on the rate of transmission may be much less than anticipated. We acknowledge the expert assistance of Raymond Paru, Henry Dagoro, Tiendepi Mininarowa, Walaf Sakel, Rhoda Espina, Wilfred Peter, Nicky Gibson, Jack Pinger, Meza Ginny and Joe Paino. We thank Dr Leonard Kaupa for permission to carry out studies in Goroka Hospital. We thank Dr Richard Hayes and Gillian Maude for statistical help and Drs Peter Heywood, Allan Saul and Chris Dye for support and advice. This project was supported by the Wellcome Trust and the US Agency for International Development. T. Burkot was supported by the The US Army Medical Research and Development Command. The project was approved by the Medical Research Advisory Committee of Papua New Guinea which acts as the National Ethical Clearance Committee for Papua New Guinea. REFERENCES

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