Prospective risk of morbidity in relation to malaria infection in an area of high endemicity of multiple species of Plasmodium.

In an area of Papua New Guinea with high prevalence of Plasmodium falciparum (39.6%), Plasmodium vivax (18.3%), and Plasmodium malariae (13.8%), cross-sectional analysis found P. falciparum infection to be independent of the other species despite heterogeneities in transmission. Plasmodium vivax and P. malariae infections were negatively correlated. Plasmodium malariae infection was positively associated with homologous infection four months previously and with prior P. falciparum, but not P. vivax infection. There were no other indications that any Plasmodium species protected against heterologous infection. Prospective analysis of health-center morbidity supported the idea that P. malariae infection protects against disease, but indicated greater protection against non-malaria than P. falciparum-associated fevers. Plasmodium vivax appeared to protect against P. falciparum disease but not against other forms of morbidity. Covariate adjustment had considerable effects on estimated relationships between species, and confounding variables may account for many differences among reports of inter-species interactions in human malaria.

Associations of peak shifts in age--prevalence for human malarias with bednet coverage.

Effects of bednet coverage (C) on prevalence of malaria were analysed using data from 1990-92 from 9 Papua New Guinean villages. Effects of coverage varied by age, resulting in a shift in age of peak prevalence from 4.7 (C = 0%) to 11.6 (C = 100%) years for Plasmodium falciparum, from 3.4 to 4.9 years for P. vivax and from 11.0 to 16.8 years for P. malariae. In small areas with no bednets the age distribution of P. falciparum parasitaemia was like that of a holoendemic area. Where coverage was complete the pattern corresponded to mesoendemicity. Thus, protracted use of bednets can result in profound changes in the endemicity of malaria even when coverage is incomplete and without insecticide treatment. Average entomological inoculation rates (EIRs) estimated from indoor landing rates on individuals without bednets were 35, 12 and 10 infectious bites per person per annum for P. falciparum, P. vivax and P. malariae, respectively. Logistic regression analyses indicated that the EIR estimate for P. falciparum was related to prevalence of this species independently of effects of bednet coverage. However, the recent EIR still accounted for much less variation than did the bednets. A similar pattern was seen for P. malariae, while there were no significant relationships between the recent EIR and the parasite positivity for P. vivax. It is concluded that short-term variations in inoculation rate are not important determinants of parasite prevalence in this population.

Age- and species-specific duration of infection in asymptomatic malaria infections in Papua New Guinea.

The burden and duration of asymptomatic malaria infections were measured in residents of the malaria endemic village of Gonoa, Madang Province, Papua New Guinea. Plasmodium falciparum, P. vivax and P. malariae infections in people aged 4 years to adulthood were compared. Frequent sampling at 3-day intervals for up to 61 days allowed assessment of individual episodes of infection. Statistical assessment of P. falciparum detection revealed a periodicity consistent with synchronous replication of this species over periods up to 27 days. The duration of P. falciparum episodes was longer across all age groups than that of P. vivax and P. malariae. A trend for decreasing duration with age was also noted in data from each species. This was most prominent in P. falciparum infections: median duration in 4-year-olds was > 48 days compared with a median between 9 and 15 days in older children and adults. The results are consistent with the slow acquisition of immunity to antigenically diverse Plasmodium populations and suggest a faster rate of acquisition to P. vivax and P. malariae than to P. falciparum.

Resistance of Plasmodium falciparum malaria to amodiaquine, chloroquine and quinine in the Madang Province of Papua New Guinea, 1990-1993.

The in vivo response of Plasmodium falciparum parasites to amodiaquine or chloroquine was assessed in children with symptomatic malaria attending different health facilities in the Madang area. Among the 27 subjects who were completely followed up, 4 (15%) were infected with parasites fully susceptible and 23 (85%) with parasites exhibiting some degree of resistance. Out of the latter group, 52% were of RI level, 26% RII and 22% RIII. 14 subjects out of 42 (33%) failed to clear their parasitaemia by day 7 and 92 out of 134 (69%) had persistent or recrudescent parasitaemia at day 21. The level of in vivo resistance was similar for amodiaquine and chloroquine. 86% of the isolates tested in vitro showed resistance to amodiaquine, 86% to chloroquine and 7% to quinine. In ten years the prevalence of resistant isolates in vivo has increased from 47% to 85%. Of more concern is the shift from RI level of resistance to RII and RIII: the proportion of resistant strains that were RI dropped from 90% to 52% over the ten-year period. To determine if the standard antimalarial regimens are still appropriate, there is a need not only to assess the level of parasite resistance but also the prevalence of treatment failure in different parts of Papua New Guinea.

PIP: The in vivo response of Plasmodium falciparum parasites to amodiaquine and chloroquine was assessed in children 1-9 years of age with symptomatic malaria recruited from health centers in Papua New Guinea's Madang area. Among the 27 children who were completely followed up, 4 (15%) were infected with fully susceptible parasites; in the remaining 23 cases (85%), there was some degree of resistance. 52% of parasites in the latter group were RI level, 26% RII, and 22% RIII. There was no correlation between level of resistance and age. 14 out of 42 children (33%) failed to clear their parasitemia by day 7 and 92 out of 134 (69%) had persistent or recrudescent parasitemia at day 21. Both amodiaquine and chloroquine had similar levels of in vivo resistance. 86% of isolates tested in vitro showed resistance to amodiaquine, 86% to chloroquine, and 7% to quinine. In 10 years, the prevalence of resistant isolates in vivo has increased from 47% to 85%. Of particular concern is the finding that the proportion of resistant strains that were RI dropped from 90% to 52% over this decade. The increase of resistance is attributed to indiscriminate use of 4-aminoquinolines in all cases of fever. Needed, to assess whether standard antimalarial regimens are still appropriate, is a review of treatment failure in different parts of Papua New Guinea.

The epidemiology of malaria in the Wosera area, East Sepik Province, Papua New Guinea, in preparation for vaccine trials. I. Malariometric indices and immunity.

The epidemiological features of malaria were studied through seven community-based surveys in a population of 4000 in the Wosera area, East Sepik Province, Papua New Guinea. Prevalence of parasitaemia (all species, all ages) was 60%. Plasmodium falciparum was the predominant species in all surveys (55%), followed by P. vivax (25%) and P. malariae (20%). The highest prevalence for asexual forms of P. falciparum occurred in the 5-9-year age group, whereas P. falciparum gametocytaemia and P. vivax parasitaemia were observed most frequently in the 1-4-year age group and P. malariae in the 10-15-year age group. Mean densities of all species decreased with age except for that of P. malariae, which was lower in children aged < 1 year than in those aged 1-4 years. The prevalence of enlarged spleen was 57% in children and 10% in adults and closely matched the corresponding age-related parasite rate. Seroprevalence of antibody to the major merozoite surface antigen 2 rapidly increased with age, with > 90% of individuals older than 5 years being positive. Malariological indices showed irregular changes over time but there was no clear-cut seasonal pattern. The geographical distribution of these indices and immune responses was not uniform within the study area. Bednet use and drug consumption were negatively correlated with malariometric indices. Identification of significant temporal and local variations in malaria endemicity is important for the design and evaluation of intervention studies, including field trials of an antimalarial vaccine.

The use of the polymerase chain reaction for more sensitive detection of Plasmodium falciparum.

The prevalence of Plasmodium falciparum in children and adults living in a malaria-endemic area in Papua New Guinea was determined by microscopy and by polymerase chain reaction (PCR). The sensitivity of detecting P. falciparum infections increased two-fold with PCR. Undetected infections by microscopy were more frequent in adults (including adolescents) than in children. Detecting this subpatent parasitaemia by PCR resulted in an equal P. falciparum prevalence in children and adults; in children the parasitaemia rate increased from 32% to 48% and in adults from 23% to 47%. In more than 50% of all blood samples positive for P. vivax and P. malariae an underlying P. falciparum infection remained undetected by microscopy. The introduction of PCR has opened up new possibilities in malaria diagnosis and research.

Relationships between Plasmodium falciparum infection and morbidity in a highly endemic area.

A total of 736 outpatients diagnosed as having malaria using clinical criteria at a health centre in a highly endemic area of Papua New Guinea were investigated parasitologically. Plasmodium falciparum-attributable fractions were determined using a logistic regression model to compare parasite densities in cases with those of healthy individuals in community surveys. Thirty-seven percent of presumptive cases were found to have raised P. falciparum parasitaemia. This corresponds to an average reporting rate for the population of 0.53 attributable episodes per annum. Whilst the maximum prevalence of parasitaemia in the community was in children aged 5-9 years, the maximum age-specific incidence of attributable cases at the outpatient clinic was 2 cases per annum in the 2- to 4-year-old age group. The procedure for estimating attributable fractions makes it possible to compare morbidity rates between age groups, and to examine how the relationship between morbidity risk and parasite density changes with age, without diagnosing individual episodes. The average tolerance of parasites in an age group was measured by considering the level of parasitaemia associated with a given risk of malaria-attributable morbidity. In contrast to anti-parasite immunity, tolerance of parasites declines with age since at parasite isodensity the probability of being symptomatic increases with age.

Malaria: how useful are clinical criteria for improving the diagnosis in a highly endemic area?

To assess the validity of clinical criteria, we investigated 2096 outpatients diagnosed as malaria cases by nurses at a rural health subcentre in a highly endemic area of Papua New Guinea. 73% of the children < 10 years old had a positive blood slide for any species of Plasmodium and 32% had > or = 10,000 P. falciparum parasites per microL. For adults the frequencies were 51% and 9%, respectively. Stepwise logistic regression identified spleen size, no cough, temperature, no chest indrawing, and normal stools as significant predictors for a positive blood slide in children; no cough and normal stools predicted a positive blood slide in adults. Fever, no cough, vomiting, and enlarged spleen were significant predictors for a P. falciparum parasitaemia > or = 10,000/microL in children; in adults the only predictor was vomiting. In children the association of no cough and enlarged spleen had the best predictive value for a positive blood slide, and a temperature > or = 38 degrees C had the best predictive value for a P. falciparum parasitaemia > or = 10,000 microL. In adults, no major symptom had a good predictive value for a positive blood slide but vomiting had the best predictive value for a P. falciparum parasitaemia > or = 10,000/microL. When microscopy is not available, these findings can help in areas of high endemicity to determine which patients with a history of fever are most likely to have malaria and, more importantly, for which patients another diagnosis should be strongly considered.

Dynamics of malaria parasitaemia associated with febrile illness in children from a rural area of Madang, Papua New Guinea.

Active community and self-reporting surveillance techniques have been used to describe the dynamics of febrile illness and associated malaria infection in children aged 2 to 15 years from a rural area of Madang Province, Papua New Guinea (PNG). Both history of fever and fever in association with parasitaemia appeared to be reliable indicators of malaria morbidity in this endemic area. Parasite density was observed to be a major determinant of mild malarial disease at both the population level and within an individual. Age-specific prevalence of febrile illness correlated with age-specific patterns of parasite density but not of parasite prevalence. Seasonal changes in fever incidence correlated with parasite density. The transition from afebrile to febrile state within an individual was generally associated with an increase in parasite density. Surveillance and self-reported febrile cases (which differ in severity on the basis of the perceived need for treatment) could be distinguished on the basis of parasite density. Thus surveillance techniques divide clinical malaria in rural PNG into 'mild' and 'very mild' forms. The age-specific pattern of decline of prevalence of malaria-associated febrile illness and parasite density is best explained by induction of strain-specific anti-disease immunity upon infection with a given strain of Plasmodium falciparum. The fever threshold in self-reporting febrile cases was seen to decrease with age and can be explained by an age-specific decline in anti-toxic immunity.

Resistance of Plasmodium falciparum to amodiaquine in Papua New Guinea.

The sensitivity of Plasmodium falciparum to amodiaquine was assessed in children in the Madang region of Papua New Guinea. Fifty-four tests in vivo were carried out and 19 (35%) of these showed resistance at the RII or RIII level. Twenty-two of the 43 isolates tested showed some degree of resistance to amodiaquine in vitro. A negative correlation between the age of the patient and apparent resistance of the parasite in vivo was observed. There was no correlation between resistance in vivo and in vitro.

Gametocyte sex ratios as indirect measures of outcrossing rates in malaria.

The frequency of recombination between unlike genotypes is central to understanding the generation of genetic diversity in natural populations of malaria. Here we suggest a way of investigating the problem which could complement conventional biochemical approaches to the population genetics of malaria. Sex allocation theory is one of the most successful areas of evolutionary biology. A well-supported prediction is that progressively less female-biased sex ratios are favoured with more outcrossing; equal numbers of males and females being evolutionarily stable in randomly mating outbred populations. We present a simple game theory model to support the idea that outcrossing rates in malaria will be correlated with the sex ratio of gametocytes in the peripheral blood of vertebrate hosts. Blood films from epidemiological surveys and culture-adapted isolates from Madang Province, Papua New Guinea, were used to estimate average gametocyte sex ratio of Plasmodium falciparum in the area. The geometric mean proportion of males in the population was 0.18 (95% confidence limits: 0.15-0.22). From our model, we estimate that, on average, 36% of zygotes are the result of outcrossing. This estimate assumes that most microgametes released following exflagellation are capable of fertilization. If, on average, fewer than about 70% of microgametes are capable of fertilization (as is the case in at least one other species of Plasmodium), the observed sex ratio would be consistent with between zero and 36% of zygotes being the result of outcrossing. These estimates suggest that there is usually a numerically dominant genotype in the gametocyte population in a blood meal, and that a considerable amount of selfing is occurring in P. falciparum populations in the Madang region, even though it is an area of intense year-round transmission.

Ineffectiveness of amodiaquine against Plasmodium falciparum malaria in symptomatic young children living in an endemic malarious area of Papua New Guinea.

The standard in vivo 7-day test of drug resistance was conducted on 83 children with symptomatic P. falciparum infection, using a full 3-day course of amodiaquine. All children were living in an endemic malarious area of the East Sepik Province of Papua New Guinea. Analysis of blood amodiaquine levels by an ELISA method showed increased blood amodiaquine concentration with progressive days of treatment. By day 7 of the study period parasites had cleared in 68 (82 per cent) of the children while 15 (18 per cent) had persistence or recrudescence of P. falciparum. Of these 15 resistant cases, four were R1 resistant (early recrudescence), seven were R2, and four were R3. This is a higher proportion of R2/R3 resistance than has usually been reported in Papua New Guinea. In vivo amodiaquine resistance declined significantly with increasing age of the child. Resistance was found to be more common in children with low weight for age: this possible association requires further investigation in the study area, which has one of the highest rates of malnutrition in Papua New Guinea.

Human host selection by anophelines: no evidence for preferential selection of malaria or microfilariae-infected individuals in a hyperendemic area.

Host selection among humans by Anopheles punctulatus was studied in an area of Papua New Guinea endemic for malaria and filariasis. Blood films were made from the stomach contents of freshly engorged mosquitoes found resting on the walls of houses in which the parasite status of the occupants was known. Engorgement rates on humans were non-random but could not be consistently related to the parasite status of individuals in the houses for either malaria or filaria. In some households, anophelines preferentially fed on parasitaemic individuals while in other households aparasitaemic individuals were significantly more often selected. This finding is believed to reflect the fact that malaria and filarial infections in this endemic area are predominantly asymptomatic. There were no significant differences in axillary temperatures between malaria or microfilariae positive or negative individuals.