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Keywords:

  • falciparum malaria;
  • malaria transmission;
  • gametocytes;
  • anaemia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary We investigated the relationship between selected host haematological and parasitological parameters and the density and infectivity of Plasmodium falciparum gametocytes. 143 individuals (age range 1–62 years) attending an outpatient clinic in Farafenni, The Gambia, who had peripheral blood gametocytaemia were recruited (mean gametocyte density 123.7/μl, range 5–17,000/μl). Of the parameters measured, packed cell volume (PCV), reticulocyte count (RetC) and lymphocyte count (LyC) were significantly associated with gametocyte density (r=− 0.17, P < 0.05; r= 0.21, P < 0.01; r= 0.18, P < 0.05, respectively). Data from membrane feeding experiments in which 15 or more mosquitoes were dissected showed that 60.7% (53/87) of gametocyte carriers infected one or more mosquitoes. Gametocyte density was strongly correlated with transmission success (TS) (r= 0.3, P < 0.005) and, in successful infections, with both mosquito prevalence (MP) (r= 0.36, P < 0.005) and mean oocyst burden (MOB) (r= 0.65, P < 0.0001). None of the other factors measured were significantly associated with any of these indices in bivariate analysis. Regression modelling showed that both gametocyte density and PCV were positively associated with gametocyte carrier infectivity to mosquitoes (LRχ2= 100.7 and 47.2, respectively) and, in successful infections, with MOB (β= 0.16, t = 4.9, P < 0.001; β= 0.02, t = 2.3, P < 0.05, respectively). The positive association with PCV suggests that blood meal quality influences infection probably as a nutritional requirement, however, as this effect was most apparent at high gametocyte densities, its epidemiological significance is questionable. Though the haematological parameters associated with gametocyte density are a direct consequence of asexual infection, they may also represent an adaptive mechanism for optimization of sexual stage development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Infection of the red blood cells with the asexual forms of the human malaria parasite, Plasmodium falciparum, results, predictably, in a number of haematological changes. One extreme manifestation is severe anaemia, a major cause of childhood death in Africa ( Greenwood et al. 1991a ). Anaemia is an inevitable consequence of erythrocyte parasitization and results primarily from the destruction of parasite-infected red cells when they rupture at schizogony. It is further exacerbated by the removal of infected and uninfected cells by the spleen, the preferential invasion of reticulocytes by merozoites and dyserythropoeisis (PasVol. et al. 1980; Francis & Warrell 1993). This reduction in overall blood ‘quality’ is also associated with shortened red cell survival, peripheral blood mononuclear cell activation and high levels of circulating cytokines. The change in these parameters contributes to the clinical presentation of malaria which typically, in Africa, is most common in young children who also support the highest asexual and sexual parasite densities ( White & Ho 1992).

Whilst the effects of parasite asexual infection on the human host are well documented, little is known of its effects on gametocytogenesis and gametocyte infectivity. It has been suggested that gametocyte production is influenced by physiological changes that accompany acute malarial infection ( Sinden 1983). Some in vitro data support this and suggest that lymphocytes, serum ( Smalley & Brown 1981), the haemolysis of infected erythrocytes ( Schneweis et al. 1991 ) and specific antibody ( Ono et al. 1986 ) can all induce gametocytogenesis.

Data on the effect of asexual P. falciparum infection on the infectivity of gametocytes are limited. In nonhuman malaria infections, asexual infection has been shown to abrogate the infectivity of the sexual stages as a result of the low haematocrit of the bloodmeal or as a consequence of circulating cytokines such as TNF-α and IFNγ ( Dearsley et al. 1990 ; Naotunne et al. 1991 ; Motard et al. 1993 ). The latter have also been shown to reduce the infectivity of P. vivax during natural infections (Mendis et al. 1989 ) and of P. falciparum when added to membrane feeding experiments ( Naotunne et al. 1991 ).

Our study used naturally infected gametocyte carriers and the membrane feeding assay to investigate the effect of asexual parasite densities and selected host haematological parameters on the density of gametocytes and on their ability to transmit infection to mosquitoes.

Methodology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study patients

The study was conducted at the Medical Research Council (MRC) field laboratory at Farafenni, The Gambia, approximately 200 km east of the capital Banjul. Malaria transmission in The Gambia is confined mainly to a three-month period at the end of the rainy season. Study patients were enrolled during the transmission seasons of 1992, 1993 and 1994 at the MRC clinic in Farafenni or at the government health centres at Farafenni and at Njaba Kunda. In all these clinics, blood films of patients suspected of having malaria are routinely examined. Patients were either identified as gametocyte carriers on presentation or were treated with antimalarials and sampled for gametocytes at MRC Farafenni one week later.

Individuals positive for gametocytes (assessed by counting 100 fields of a thick blood film stained with Field's stains) and older than one year were asked to participate in the study. If consent was obtained, a 3 ml blood sample was taken by venepuncture into heparinized tubes and the sample maintained at 37 °C until membrane feeding was performed. Each day, patients were selected from those first identified as gametocyte-positive; the number of experiments conducted was typically 1–2.

Parasitological and haematological measurements

Thick blood films were stained with Fields' stain and used for both asexual and sexual parasite counts; one parasite per high power field was taken as equivalent to 500 parasites/μl ( Greenwood & Armstrong 1991b).

Red blood cell counts (RBCC), percentage reticulocyte counts (RetC), packed cell volumes (PCV), white blood cell counts (WBCC) and sickle cell tests were performed according to standard procedures (WHO 1980). Lymphocyte and neutrophil counts (LyC, NeC) were estimated by multiplying the proportion of each cell type obtained from a differential count (counting 500 white blood cells on a Giemsa stained thin film) by the WBCC. Haemoglobin concentration was measured by adding 20 μl of whole blood to 5 ml Drabkins solution (1 vial of Drabkins reagent (Sigma, Poole, UK 525–2), 0.5 ml BRIJ-35 (Sigma, 430AG-6) in 1 l distilled H20) and reading the absorption on a spectrophotometer at a wavelength of 540 nm. Samples were blood typed for ABO and Rhesus group using an agglutination test according to manufacturers instructions (Ortho Diagnostics, New Jersey).

Source of mosquitoes

'Fully fed' adult female mosquitoes were collected in villages known to have a high proportion (> 90%) of An.gambiae s.s. ( Bryan et al. 1979 ). Mosquitoes were allowed to oviposit, the eggs were collected and reared through larval and pupal stages in an insectary maintained at 28 °C ± 2 °C and 70–90% relative humidity (RH).

Membrane feeding of mosquitoes

Groups of 50, 3–5-day-old F1 female mosquitoes were counted into small paper cups covered with mosquito netting. 1 ml aliquots of blood from gametocyte positive patients were placed in glass feeders with a Parafilm® (American Can Co., Connecticut) membrane maintained at 37 °C. Mosquitoes were allowed to feed for 15 min, after which unfed or partially fed mosquitoes were removed. Fully fed mosquitoes were maintained in the insectary with a constant supply of 10% sucrose for 7 days. On day 8 after feeding, mosquitoes were dissected into normal saline, their midguts were stained with 2% mercurochrome and examined for oocysts.

Statistical analysis

Statistical analysis was performed using EPI-Info 6.0 (CDC, Atlanta & WHO, Geneva) and STATA (STATA Corp, Texas) statistical packages. Significance was tested at the 5% level. Spearmans rank correlation was used to investigate whether variables were associated with gametocyte density. The χ2 test with Yates continuity correction was used to compare proportions with respect to transmission success (TS). The students T-test or anova were used to compare means of mosquito prevalence (MP; the percentage of mosquitoes infected) and mosquito oocyst burden (MOB; the mean number of oocysts per midgut in infected mosquitoes). Poisson regression analysis was used to model gametocyte infectivity. The number of oocyst-positive mosquitoes was used as the dependent variable with the number of mosquitoes dissected as a measure of exposure. This method had the advantage in that both positive and negative infections and experiments in which low numbers of mosquitoes were dissected could be included in the analysis. Standard linear regression was performed with both MP and MOB as dependent variables.

Ethical considerations

The study and all procedures involved were approved by the Scientific Co-ordinating Committee of the Medical Research Council and The Joint Gambia Government – Medical Research Council Ethical Committee. Informed consent was obtained from all patients or their guardians. Treatment of patients according to standard practice (The Gambia Government treatment guidelines) was carried out by trained MRC and health centre staff.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Parasitological data

143 gametocyte carriers were enrolled in the study and categorized by age group ( Table 1); 53% of were male and their ethnic group composition mirrored that of the total population (data not shown). The highest gametocyte count recorded was 17 000 gametocytes/µl seen in a 7-year-old girl. 70 gametocyte carriers (49%) had asexual parasites at presentation and there was a trend for the prevalence of asexual parasites to decrease with age (χ2= 8.1, P= 0.017; Table 1).

Table 1.   Parasitological data on 143 gametocyte carriers by age group Thumbnail image of

Haematological data

Proportions and mean values of haematological parameters in the study population are presented in Table 2. As expected, the most severe haematological disruption was seen in the youngest age group and was characterized by low PCV, RBCC, Hb levels and high WBCC and RetC levels.

Table 2.   Mean haematological measurements between age groups Thumbnail image of

Associations with gametocyte density

No significant differences in mean gametocyte densities were seen between the sexes or between age groups ( Table 1). No significant relationship between gametocyte count and the presence or the density of asexual parasites was seen. Gametocyte density was correlated significantly with PCV, RetC and LyC (r=− 0.167, P= 0.047; r= 0.212, P= 0.010; r= 0.178, P= 0.033, respectively) and with WBCC at borderline significance (r= 0.158, P= 0.066).

Gametocyte carrier infectivity

Membrane feeding experiments were performed with samples from all patients but the number mosquitoes surviving to dissection varied between tests. Only experiments in which 15 or more mosquitoes were dissected were included in the initial analysis (n= 87). At least one mosquito became infected in 53 of 87 experiments (60.9%; Table 3). In the positive experiments, the mean percentage of infected mosquitoes was 24.8% with the highest MP of 77.4% seen after feeding mosquitoes on a 60-year-old with 110 gametocytes/μl. The arithmetic MOB in successful experiments was 9.3 oocysts per gut and the highest arithmetic MOB in an individual experiment was 60.6 after feeding mosquitoes on a 7-years-old child with 17 000 gametocytes/μl. All individuals in the older age group were infectious to mos- quitoes.

Table 3.   Data on infectivity of gametocyte carriers by age group Thumbnail image of

Parasitological data and infectivity

Mean gametocyte density was significantly higher in successful than in unsuccessful experiments ( Table 4). Gametocyte density was also strongly correlated with both MP (r= 0.33, P= 0.018) and MOB (r= 0.57, P= 0.0001). No relationship was seen between the presence or density of asexual parasites with TS ( Table 4), MP or MOB (data not shown).

Table 4.   Relationship between parasitological measurements and transmission success Thumbnail image of

Haematological factors and transmission

Bivariate analysis

There were no significant differences in mean levels of haematological parameters between unsuccessful and successful experiments ( Table 5). The observation that individuals of blood group B were less infectious to mosquitoes was of borderline significance ( Table 5). Haematological measurements were redefined as abnormal or normal using standard values for each age group ( Dacie & Lewis 1975; Smith et al. 1989 ; Shaper & Lewis 1971); again there were no significant associations with TS ( Table 5), MP or MOB (data not shown).

Table 5.   Relationship between haematological parameters and transmission success Thumbnail image of
Multivariate analysis

Poisson regression was used to model gametocyte infec- tivity. Both gametocyte count and PCV were associated positively with mosquito infectivity (LRχ2= 100.7, d.f. = 1, P < 0.0001 & LRχ2= 47.2, d.f. = 1, P < 0.0001, respectively). Similarly in a linear regression model, restricted to successful infections with 15 or more mos-quitoes dissected, with mean oocyst count (log adjusted) as the dependent variable both gametocyte count and PCV were positively associated (β= 0.16, t = 4.9, P= 0.001; β= 0.02, t = 2.3, P= 0.028, respectively). The effect of PCV on both MP and MOB is shown in Figures 11 and 2, in which PCV has been coded as less than or greater than 30%.

image

Figure 1.  The relationship between the percentage of mosquitoes infected and gametocyte density at arbitrary high (> 30%▪) PCV levels.

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image

Figure 2.  The relationship between the mean oocyst burden of mosquitoes infected and gametocyte density at arbitrary high (> 30%▪) and low (< 30%□) PCV levels.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We investigated the effect of host haematological and parasitological parameters on the density of gametocytes and their infectivity to mosquitoes. Our in vivo data show that the density of gametocytes correlated with a number of haematological parameters, packed cell volume, lymphocyte count and reticulocyte count, which have also been associated with gametocytogenesis in vitro. Products from lysed erythrocytes induce gametocytogenesis in cultured parasites ( Schneweis et al. 1991 ) and these products might be expected in individuals with anaemia as indicated by a low PCV. Similarly, peripheral blood mononuclear cells from infected Gambian children stimulated gametocytogenesis in cultured isolates ( Smalley & Brown 1981). This may be extrapolated to those individuals with high LyC and WBCC though gametocytogenesis is more likely to be influenced by the soluble products released by these cells and not by their overall number. Increased gametocytogenesis has also been observed in culture systems enriched with reticulocytes ( Trager & Gill 1992) and this may correspond to individuals with high RetC.

The observed changes in these haematological parameters can be expected as a consequence of infection with asexual parasites. Whether gametocytogenesis is induced as a response to these changes or if it is an adaptive measure of the parasite to take advantage these changes as optimal conditions for sexual stage development is not clear; gametocytogenesis maybe simply programmed to occur after so many cycles of asexual replication.

The results from experimental infections showed that blood from 60.5% of gametocyte carriers was infectious to mosquitoes; a figure comparable with data from other studies using membrane and direct feeding in malaria-endemic areas ( Muirhead-Thomson 1957; Rutledge et al. 1969 ; Graves et al. 1988 ; Githeko et al. 1992 ; Tchuinkam et al. 1993 ; Boudin et al. 1993 ). Also comparable with the other membrane feeding studies was the observation that gametocyte density correlated with TS, affecting both the MP and MOB. Interestingly, the range and the mean number of oocysts in infected mosquitoes appear to be much higher in this study compared to others ( Tchuinkam et al. 1993 ; Boudin et al. 1993 ). A possible reason for this is that first-generation female progeny of local mosquitoes were used in this study compared with established laboratory strains in the other studies. The use of laboratory strains of mosquitoes may thus underestimate the number of infectious individuals or overestimate the effect of a particular transmission-blocking intervention.

Also consistent with other studies were the observations that no one individual infected 100% of mosquitoes, and that low densities of gametocytes were highly infectious and high densities of gametocytes were not infectious. This paradox of infectivity has been noted in most studies assessing human malarial infectivity to mosquitoes ( Jeffery & Eyles 1955; Rutledge et al. 1969 ; Gamage-Mendis et al. 1991 ; Tchuinkam et al. 1993 ); infectivity maybe dependent on the elusive ‘quality’ not quantity of gametocytes ( Boyd 1949).

Data on the of age of gametocyte donor and parasite infectivity are too few to draw any strong conclusions. Interestingly, all the individuals in the older age group included in the analysis were infectious (8/8) and, including all experiments, 80% (8/10) of individuals older than 20 infected one or more mosquitoes. Only 1 of this latter group of adults had presented with asexual malaria, indicating asymptomatic gametocyte carriage by adults. It has been suggested that adults contribute less to the infectious reservoir of the parasite due to lower prevalence and density of parasites (asexual and sexual) and increased levels of transmission-blocking immunity ( Githeko et al. 1992 ; Boudin et al. 1993 ). However, observations with P. vivax gametocyte carriers showed that while gametocyte density decreased with age, infectivity ‘per gametocyte’ increased markedly ( Gamage-Mendis et al. 1991 ). The evidence from this study suggests a low prevalence of natural transmission-blocking immunity in all age groups. Laboratory studies are underway to investigate the sero-reactivity of samples from the gametocyte carriers to sexual stage antigens.

One intriguing finding is the tendency for individuals with blood group B to be less infectious than those of either blood group O or A. A similar observation was made in experiments performed in Cameroon ( Tchuinkam et al. 1993 ) and when the two data sets are combined the effect is more pronounced (χ2= 6.7, P < 0.05). Only 46% of blood group B individuals were infectious (n= 48) compared with 64% for group A (n= 59) and 67% for group O (n= 110). Whether this is another biological adaptation of the parasite to the relative proportion of blood groups in West Africa ( Facer & Brown 1979) or a chance finding is not clear but further investigation is warranted.

The principle reason for measuring haematological and parasitological parameters was to obtain an indication of the severity of the clinical disease in a particular gametocyte carrier and its subsequent influence on the transmission of the parasite. Apart from gametocyte density only PCV had an association with the outcome of transmission. This has been shown previously with P. gallinaceum ( Rosenberg et al. 1984 ) and probably reflects the requirement of protein for parasite development ( Hurd et al. 1995 ). Whether this interaction has any epidemiological significance is not clear. Gametocyte densities in the general population are low ( Taylor & Read 1997) and the effect of PCV is most pronounced at higher densities (Figures 11 and 2). Also, after infection the return of PCV levels to normal is slow, 21–28 days in children ( Amukoye et al. 1997 ) and, even allowing for the long developmental period and half life of P. falciparum gametocytes, this maybe too long for a positive effect of the interaction to be observed.

Given our observations and the undoubted success of P. falciparum, its gametocytes are clearly robust enough to survive the potentially harmful effects of asexual infection and to optimize infectivity. Whilst the infectivity of gametocytes of other malarias can be affected by asexual infection through cytokines or low haemoglobin levels, these nonspecific effects do not appear to affect P. falciparum.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We wish to acknowledge the expert assistance of Nfansu Colley, Hamoro Camara, John Camara, Gibril Bass, Olaf Muller, Umberto D'Alessandro and Batch Cham at the Farafenni field station. Many thanks to Pate Makolo, Mamadou Jallow and Gallo Kebbeh for performing the mosquitoes catches and to Jo Armstrong-Schellenberg for statistical advice.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Methodology
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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