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Am. J. Trop. Med. Hyg., 78(3), 2008, pp. 442-448
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Increased Plasmodium falciparum Gametocyte Production in Mixed Infections with P. malariae

J. Teun Bousema*, Chris J. Drakeley, Petra F. Mens, Theo Arens, Rein Houben, Sabah A. Omar, Louis C. Gouagna, Henk Schallig, AND Robert W. Sauerwein
Department of Medical Microbiology, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands; Kenya Medical Research Institute, Nairobi, Kenya; Human Health Division, International Centre of Insect Physiology and Ecology, Nairobi, Kenya; Unité de Recherche 016, Institut de Recherche pour le Développement, Ouagadougou, Burkina Faso


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmodium falciparum and P. malariae occur endemically in many parts of Africa. Observations from malariotherapy patients suggest that co-infection with P. malariae may increase P. falciparum gametocyte production. We determined P. falciparum gametocyte prevalence and density by quantitative nucleic acid sequence-based amplification (QT-NASBA) after antimalarial treatment of Kenyan children with either P. falciparum mono-infection or P. falciparum and P. malariae mixed infection. In addition, we analyzed the relationship between mixed species infections and microscopic P. falciparum gametocyte prevalence in three datasets from previously published studies. In Kenyan children, QT-NASBA gametocyte density was increased in mixed species infections (P = 0.03). We also observed higher microscopic prevalences of P. falciparum gametocytes in mixed species infections in studies from Tanzania and Kenya (odds ratio = 2.15, 95% confidence interval = 0.99–4.65 and 2.39, 1.58–3.63) but not in a study from Nigeria. These data suggest that co-infection with P. malariae is correlated with increased P. falciparum gametocytemia.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmodium falciparum and P. malariae are malaria species that occur endemically in many parts of sub Saharan Africa. Mixed infections with both species are usually observed in a small proportion of persons in cross-sectional studies (2–14%),1–4 although their cumulative prevalence has been documented at 20–50% in longitudinal studies conducted over 1–2 years.2–4 The co-occurrence of P. falciparum and P. malariae is often higher than would be expected on the basis of their individual parasite prevalence,5–8 and one parasite species may influence the infection dynamics of the other. The biologic and subsequent clinical interactions are therefore of interest, especially in light of vaccination trials specifically targeted at P. falciparum.

In mixed infections, the presence of P. malariae parasites can influence the disease manifestation of P. falciparum with a reduction in disease severity9 and a lower peak parasitemia,10 possibly as a consequence of heterologous immunity.11,12 Observations from malariotherapy patients indicate that co-infection with P. malariae may increase P. falciparum gametocytemia.13 If this increased gametocyte production is manifested in co-infections in field settings, this could have implications for the spread of malaria and malaria control. Predictably few field studies have addressed this issue: most clinical studies focus on P. falciparum mono-infections and use co-infection with other malaria species as an exclusion criterion. A study of 5,682 symptomatic patients in Thailand found that P. falciparum gametocytes were less common in mixed infections with P. vivax.14 A small cross-sectional study in 25 households in Mozambique suggested that P. falciparum gametocytes may also be less common in the presence of P. malariae.1 We examined the relation between P. falciparum gametocytemia and mixed infection with P. malariae in four separate studies. We compare P. falciparum gametocyte prevalence and density assessed by molecular methods after anti-malarial treatment of Kenyan children naturally infected with either P. falciparum mono-infection or P. falciparum and P. malariae mixed infection. In addition, we present analyses of the relationship between mixed species infections and microscopic P. falciparum gametocyte prevalence in three large datasets from previously published studies conducted in Tanzania, Kenya, and Nigeria.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gametocytemia and mixed species infections after antimalarial drug treatment. The study assessing P. falciparum gametocyte prevalence and density after antimalarial treatment was conducted from October to December 2004 in Mbita, a rural village on the shores of Lake Victoria in the Suba District of western Kenya. Transmission intensity is high and perennial in the study area (entomologic inoculation rate approximately six infectious bites per person per month)15 with P. falciparum as predominant parasite species accounting for more than 95% of the clinical malaria cases.16 Patients with P. falciparum mono-infection were enrolled as part of a larger drug sensitivity study that was reported previously (Clinical Trials registration no. ISRCTN31291803 available from http://www.controlled-trials.com/ISRCTN31291803).17 Children 6 months to 10 years of age with a temperature > 37.5C° measured by earthermometer or a history of fever within the last 48 hours and with P. falciparum mono-infection at a density between 500 and 100,000 parasites/µL were eligible for recruitment. Exclusion criteria were inability to take drugs orally, known hypersensitivity to any of the drugs given, reported treatment with antimalarial chemotherapy in the past two weeks, evidence of chronic disease or acute infection other than malaria, and domicile outside the study area and signs of severe malaria. Children with P. falciparum and P. malariae mixed infection were enrolled in the study on the basis of the same criteria but no minimum parasite density was used as an inclusion criterion. All subjects included in the analyses were treated with sulfadoxine-pyrimethamine (SP; Fansidar®; Hoffmann LaRoche, Basel, Switzerland) plus amodiaquine (AQ), 10 mg/kg, once a day for three days (Camoquine®; Pfizer, Dakar, Senegal) and subsequently followed for 28 days. The study protocol (SSC no. 791) was reviewed and approved by the Scientific Steering Committee and Ethical Review Committee of the Kenya Medical Research Institute, Nairobi, Kenya.

Microscopy and molecular parasite detection. Giemsa-stained blood smears were screened for asexual parasites and gametocytes at enrollment and on days 3, 7, 14, and 28 after treatment. Slides were declared negative if no parasites were observed in 100 microscopic fields, each containing approximately 15–20 leukocytes; asexual parasites and gametocytes were counted against 200 and 500 leukocytes, respectively. Conversion to parasites/microliter was made using a conversion rate of 8,000 leukocytes/µL. Parasite detection by quantitative nucleic acid sequence-based amplification (QT-NASBA) was done for a random selection of P. falciparum mono-infections with complete follow-up (47 of 127)17 and for all P. falciparum plus P. malariae mixed infections (21 of 21). Nucleic acids were extracted from 50-µL finger prick blood samples by the method described by Boom and others.18 Plasmodium falciparum QT-NASBA was performed on a NucliSens EasyQ analyzer (bioMérieux, Boxtel, The Netherlands) as described elsewhere for Pfs25 mRNA.19 Pfs25 mRNA is only expressed in stage V P. falciparum gametocytes,20 and the Pfs25 QT-NASBA has a detection limit of 20–100 gametocytes/mL. Nuclisens Basic kits (bioMrieux) é were used for amplification according to the manufacturer’s instructions at a KCl concentration of 80 mM. A standard dilution series of in vitro cultured mature NF54 gametocytes21 was included in each run to ascertain gametocyte density. The presence of P. malariae parasites in mixed infections was confirmed by detecting P. malariae-specific 18S ribosomal RNA using QT-NASBA.22

Data analyses. Nonparametric Wilcoxon rank sum tests were used to test differences between groups for statistical significance in case of continuous variables; chi-square tests were used for dichotomous variables. Multiple logistic regression models with generalized estimating equations (GEEs) were used to test the influence of P. falciparum plus P. malariae mixed infection on Pfs25 QT-NASBA gametocyte prevalence. A similar procedure was carried out using GEEs for Pfs25 QT-NASBA gametocyte density. Estimates were adjusted for potential confounding factors (i.e., age, treatment outcome, microscopic asexual parasite density at enrollment, and fever at enrollment) and a random effect was included in the models to show correlations within persons. To quantify the influence of co-infection with P. malariae on P. falciparum gametocyte densities during follow-up, we determined the area under the curve (AUC) of Pfs25 QT-NASBA gametocyte density versus time.23,24 This measure incorporates both the magnitude and the duration of transmission potential and was described by Mendez and others.24 The AUC from days 0 to 42 was calculated as AUC = [(3 - 0) x (g0 + g3)/2 + (7 - 3) x (g3 + g7)/2 + (14 - 7) x (g7 + g14)/2 + (28 - 14) x (g14 + g28)/2]/28 where gd represents Pfs25 QT- NASBA gametocyte density on day d. Gametocyte negative samples were included as zeroes. The measure was scaled by 28 so that it represents AUC per day and this was transformed by log10 for comparisons. Statistical analyses were performed using procedures available in SPSS version 12.0 (SPSS Inc., Chicago, IL) and Stata version 8.0 (Stata Corporation, College Station, TX).

Analyses on data from three previously conducted studies. Data from three studies were analyzed on the basis of the availability of information on the prevalence of P. falciparum asexual parasites and gametocytes and P. malariae asexual parasites.

The first dataset was obtained from a series of cross-sectional studies in six altitude transects (150–1,800 meters) in the Kilimanjaro and Tanga regions in northern Tanzania.25 Cross-sectional surveys in individuals 6 months to 45 years of age were conducted twice in a period of 6 months; 100 microscopic fields were screened for asexual parasites of P. falciparum and P. malariae and gametocytes of P. falciparum. Asexual and gametocyte densities were recorded per 200 leukocytes and 500 leukocytes, respectively.

The second dataset was obtained from a longitudinal study in Mbita in western Kenya.26 Children 6 months to 16 years of age were screened weekly for five weeks; 100 microscopic fields were screened for asexual parasites of P. falciparum and P. malariae and gametocytes of P. falciparum.

The third dataset was obtained from the Garki dataset from 1970 and 1971, the years prior to the transmission-reducing intervention (http://www.sti.ch/research/biostatistics/downloads.html).27 In this part of the study, surveys were conducted in the general population (0–72 years of age) of villages in Garki in northern Nigeria. Every ten weeks slides were collected and 200 or 400 microscopic fields were screened for asexual parasites of P. falciparum and P. malariae and gametocytes of P. falciparum. Parasite densities were calculated as the percentage of positive fields. Sampling took place every 10 weeks.

Microscopic gametocyte density data were available for the Kenyan and Tanzanian datasets but because there was little variation in these densities (typically 16–32 gametocytes/µL), we considered it appropriate to present microscopic gametocyte prevalence data only. Although the sampling interval in the Tanzanian and Nigerian datasets was relatively large, this may not guarantee independence of observations.28 Therefore, multiple logistic regression models with GEEs were initially used to test the relation between P. falciparum gametocyte prevalence and the presence of P. falciparum and P. malariae mixed infections in all data sets. Estimates were adjusted for potential confounding factors and a random effect was included in the models to show correlations within persons. If the addition of the random effect did not improve the model, as was the case for data from Tanzania, outcomes of conventional logistic regression models were presented.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gametocytemia and mixed species infections after antimalarial drug treatment. In Mbita, Kenya, 127 P. falciparum mono-infections and 21 P. falciparum and P. malariae mixed infections were treated with SP plus AQ. The presence of P. malariae parasites was confirmed by P. malariae 18S ribosomal RNA QT-NASBA in all mixed species infections (21 of 21) and in none of the P. falciparum mono-infections (0 of 47) tested. Complete 28-day follow-up data were available for 115 P. falciparum mono-infections and 20 mixed species infections. An adequate clinical response was observed in 87.0% (100 of 115) of the P. falciparum mono-infections and 100% (20 of 20) of the mixed species infections (P = 0.13). Children with mixed species infections were significantly older than those with P. falciparum mono-infections (P = 0.007; Table 1Go). Microscopic P. falciparum gametocyte prevalence was non-significantly higher in mixed species infections (P = 0.26; Table 1Go).


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TABLE 1
Baseline characteristics of children with Plasmodium falciparum mono-infection or P. falciparum and P. malariae mixed infection*
 
Gametocyte prevalence by Pfs25 QT-NASBA was 91.1% (41 of 45) in P. falciparum mono-infections at enrollment and 90.5% (19 of 21) in mixed species infections (P = 0.93; Table 1Go). The Pfs25 QT-NASBA gametocyte prevalence at enrollment was negatively associated with age (β = –0.36, SE = 0.17, P = 0.04) and showed a weak positive association with microscopic asexual parasite density (β = 0.38, SE = 0.35, P = 0.28). On the third day after the initiation of treatment, Pfs25 QT-NASBA gametocyte prevalence was non-significantly higher in mixed species infections (P = 0.07) (Figure 1AGo) without any clear difference during the rest of follow-up. When the entire period of follow-up was considered, there was no statistically significant difference in the prevalence of gametocytes by Pfs25 QT-NASBA between P. falciparum mono-infections and mixed species infections (GEE β = 0.21, SE = 0.42, P = 0.62), after adjustment for age and asexual parasite density at enrollment. However, in Pfs25 QT-NASBA gametocyte carriers, the Pfs25 QT-NASBA gametocyte density was consistently increased in mixed species infections (Figure 1BGo). The geometric mean Pfs25 QT-NASBA density was more than two-fold higher on the third day after initiation of treatment in mixed species infections: 2.9 gametocytes/µL (95% confidence interval [CI] = 0.7–12.4) for P. falciparum mono-infections and 8.2 gametocytes/µ L (95% CI = 3.1–21.3) for mixed species infections (P = 0.11). The Pfs25 QT-NASBA gametocyte density throughout the study period was significantly higher in mixed infections than in mono-infections (GEE β = 0.38, SE = 0.17, P = 0.03) after adjustment for age and microscopic P. falciparum asexual parasite density at enrollment. Treatment outcome and the presence of fever at enrollment did not confound this relationship.


Figure 1
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  		    FIGURE 1. Plasmodium falciparum gametocyte prevalence and density determined by Pfs25 quantitative nucleic acid sequence-based amplification (QT-NASBA) in P. falciparum mono-infections and mixed infections with P. malariae. Shown are QT-NASBA gametocyte prevalence (A) and density (B) for P. falciparum mono-infections (closed triangles, broken line) and P. falciparum plus P. malariae mixed infections (open squares, solid line). Bars indicate the 95% confidence intervals.

 
The transmission potential during follow-up, quantified as the AUC of Pfs25 QT-NASBA gametocyte density versus time, was significantly higher for mixed species infections (P = 0.02; Table 2Go). The number of sampling times when gametocytes were detected by Pfs25 QT-NASBA was not significantly different between the two groups (P = 0.45), but the geometric mean Pfs25 QT-NASBA gametocyte density in Pfs25 QT-NASBA gametocyte-positive samples was hig her in mixed species infections (P = 0.03; Table 2Go).


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TABLE 2
Pfs25 QT-NASBA gametocyte carriage during follow-up for children with Plasmodium falciparum mono-infection or P. falciparum and P. malariae mixed infection*
 
Gametocyte prevalence in mixed species infections in previously conducted studies. We analyzed three datasets on the relationship between P. falciparum and P. malariae mixed infection and the prevalence of P. falciparum gametocytes. Prevalence data were available from multiple time points. Data were presented for all time points combined; the fact that multiple observations were derived from the same persons was taken into account in the statistical analysis. There was considerable variation in the strength of the association between microscopic P. falciparum gametocyte prevalence and mixed species infection (Table 3Go). We observed a borderline significant increase in P. falciparum gametocyte prevalence for mixed species infections in data from different sites in north eastern Tanzania (odds ratio [OR] = 2.15, 95% CI = 0.99–4.65) and this increase was statistically significant for data from Kenya (OR = 2.39, 95% CI =.58–3.63), after adjustment for age and concurrent microscopic asexual parasite density. In the dataset from Nigeria, the effect varied between different age groups. There was a borderline significant lower microscopic P. falciparum gametocyte prevalence in mixed species infections for persons less than 10 years of age (OR = 0.88, 95% CI 0.56–1.00), and there was a weak and non-significant higher risk in older children after adjustment for age and concurrent microscopic P. falciparum asexual parasite density (OR = 1.13, 95% CI 0.87–1.48).


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TABLE 3
Re-examination of studies detecting Plasmodium falciparum gametocytes in mono-infections and P. falciparum plus P. malariae mixed infections*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that co-infection with P. malariae is correlated with an increase in P. falciparum gametocytes. This finding is consistent in three different studies from different malaria-endemic areas, using both microscopy and molecular methods; the effect was not apparent in a fourth study.

In Kenyan children with symptomatic malaria, we observed a non-significantly higher microscopic P. falciparum gametocyte prevalence prior to treatment. Although Pfs25 QT-NASBA gametocyte prevalence was not significantly increased, Pfs25 QT-NASBA gametocyte density was consistently higher in mixed species infections throughout one month of follow-up. This finding could not be explained by differences in treatment efficacy, age, fever, or enrollment parasite density. Kenyan children with mixed species infections were more likely to respond well to treatment, were older, and had lower P. falciparum asexual parasite densities at enrollment; all factors previously associated with lower rather than higher gametocyte prevalence and density.26,29

In P. falciparum, most gametocytes that appear after treatment are likely to have been committed to sexual stage development prior to treatment. Gametocytes take 8–12 days to develop30 and the peak in gametocyte prevalence and density that is commonly seen after treatment is at least partly the result of an efflux of these sequestered gametocytes.31 Although a more pronounced release of gametocytes in the presence of P. malariae co-infection could explain our findings after antimalarial treatment, it seems more plausible that mixed species infections have a higher commitment to production of P. falciparum gametocytes that persist after treatment, without a causative role for treatment as such. This finding is also suggested by the non-significantly increased microscopic gametocyte prevalence and Pfs25 QT-NASBA gametocyte density in mixed species infections prior to treatment.

Retrospective analysis of data from previously conducted epidemiologic studies show a similar picture. In studies conducted in Kenya and Tanzania, we observed a significantly higher microscopic prevalence of P. falciparum gametocytes in mixed species infections. Because microscopy underestimates the total proportion of gametocyte carriers and only detects relatively high gametocyte densities,19 these microscopic findings can be interpreted as a higher prevalence of high density gametocyte carriage in mixed species infections.

The Nigerian Garki study did not confirm the relationship between P. falciparum gametocytemia and mixed species infections. In contrast, there was a non-significantly lower P. falciparum gametocyte prevalence associated with mixed species infection in children less than 10 years of age,1 and there was a weak and non-significant increased risk of P. falciparum gametocyte carriage in older persons with mixed species infections. This finding indicates that the relationship between mixed species infections and gametocytemia may be different under different endemicities. Despite this consideration, our findings in three independent datasets strengthen our conclusion that P. falciparum gametocyte production may be higher in the presence of P. malariae parasites. This would confirm observations from malariotherapy patients where co-infection with P. malariae stimulates P. falciparum gametocyte production.13 A possible influence in the other direction, i.e., a change in P. malariae gametocytemia in mixed species infections, could not be determined. Plasmodium malariae gametocytes were not recorded in any of the studies discussed in this report and a molecular tool to detect sexual stage parasites of other species than P. falciparum is currently unavailable.

Although three different studies show a similar relationship between mixed species infections and P. falciparum gametocytemia, our findings should be interpreted with caution. Malaria-associated leukopenia may have impaired our estimate of microscopic gametocyte density in Kenyan children with symptomatic malaria, where a standard leukocyte concentration of 8,000 cells/µL was assumed.32 Moreover, the extent of leukopenia may differ between different plasmodium species32 and could therefore confound relationships between mixed species infections and microscopically estimated asexual parasite or gametocyte densities. Leukocyte counts are, however, unlikely to have influenced the major outcome measures of this study, which were Pfs25 QT-NASBA gametocyte density and prevalence and microscopic gametocyte prevalence. In addition, none of the conducted studies were specifically designed to detect interactions between malaria species. This would ideally require longitudinal studies in the absence of antimalarial treatment. Our design hampers us to draw conclusions on causality in the observed relations. Plasmodium falciparum infections show parasitic waves that may be followed by an increased gametocyte production.33 Similarly, P. malariae parasite densities may increase when P. falciparum densities decrease.34 This may result in a simultaneous increase in concentrations of P. malariae asexual parasites and P. falciparum gametocytes, making them more likely to be detected by microscopy, without any causal relationship between the two.

If there is a causal relationship between mixed species infections and gametocyte production, it is likely to be mediated by cross-species immune responses. Mixed genotype infections have been associated with higher transmission success and higher gametocytemia in animal models.35 A similar phenomenon may play a role in mixed species infections and may indicate a response of parasites in terms of transmission potential in the presence of a competitor. Although antibody-mediated parasite immunity seems to be largely species and strain-specific, it is likely that there is a certain degree of cross-reactive immunity between species.36,37 There is evidence for heterologous protective immunity for P. falciparum induced by P. malariae9,37 and for cross-species regulation of parasite densities.12,38,39 This cross-species immunity may also influence malaria transmission.36 A higher infectivity of parasites to mosquitoes in the presence of a different malaria species was observed for simian malaria species40 and for human P. vivax and P. falciparum in Aotus monkeys.41 Although we did not determine immune responses, we hypothesize that cross-reactive antibodies may play a role in explaining our findings. Antibodies that are cross-reactive between species may increase the immune stress experienced by P. falciparum, thereby stimulating this species to invest in transmission stages. The observation from the Garki dataset that increased gametocyte prevalence is only observed in children more than 10 years of age may be related to the development of clinical immunity in this age group. Further analysis allowing for the effects of transmission intensity of both P. falciparum and P. malariae is warranted but beyond the scope of this report.

The relevance of our findings for malaria transmission depends on prevalence of mixed species infections and influence of an increase in gametocyte density on malaria transmission potential. We have recently shown a positive association between gametocyte density and the proportion of infected mosquitoes, including submicroscopic gametocyte densities.42 Thus, the higher densities of gametocytes (and the higher prevalence of high density gametocyte carriage) in mixed species infections are likely to result in a higher proportion of infected mosquitoes. The infectiousness of gametocytes in symptomatic Kenyan children was confirmed on day 14 after the initiation of treatment, with 23 of 28 children with P. falciparum mono-infections17 and 3 of 4 children with P. falciparum and P. malariae mixed infections infecting at least one mosquito. The prevalence of mixed species infections in a given population is highly variable. Plasmodium falciparum and P. malariae mixed infections may account for 6% of the P. falciparum malaria cases in western Kenya,43 but the prevalence may be much higher in longitudinal studies2–4 or in studies using species specific molecular detection techniques.1,44 In our study, we found no sub-patent P. malariae infections in children with apparent P. falciparum mono-infections although a study from Mozambique found a twofold increase in the proportion of mixed species infections using a polymerase chain reaction.1 The prevalence of P. malariae infections may further increase when P. falciparum-specific control programs are implemented,2 such as current vaccine trials.

In conclusion, our data suggest that the transmission potential of P. falciparum is increased by co-infection with P. malariae. Longitudinal studies are needed to confirm this relationship, to identify possible mechanisms, and to determine the duration and relevance of the potential increase in malaria transmission that we observe. These longitudinal studies should preferably use molecular tools to detect and quantify the different stages of parasite species in the absence of malaria treatment.

Received May 7, 2007. Accepted for publication November 19, 2007.

Acknowledgments: We thank the community of Mbita for their cooperation; and S. Kaniaru (Kenya Medical Research Institute), G. Omweri, N. Makio, P. Sawa, B. Kapesa, K. Okoth and P. Ongele (International Centre of Insect Physiology and Ecology) for their work at the clinic and in the field. We also thank the Joint Malaria Programme (a collaboration between the Tanzanian National Institute for Medical Research; Kilimanjaro Christian Medical Centre; the London School of Hygiene and Tropical Medicine, and the Centre for Medical Parasitology, University of Copenhagen) for access to the data from the Usambara Mountains.

Financial support: J. Teun Bousema is supported by The Netherlands Foundation for the Advancement of Tropical Research (W 07.05.203.00) through Poverty Related Infection Oriented Research, and Chris J. Drakeley is supported by a research fellowship in tropical medicine (#063516) from the Wellcome Trust.

* Address correspondence to J. Teun Bousema, Department of Medical Microbiology 268, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: t.bousema{at}ncmls.ru.nl Back

Authors’ addresses: J. Teun Bousema, Theo Arens, and Robert W. Sauerwein, Department of Medical Microbiology 268, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, The Netherlands, Telephone: 31–24–361–9515, E-mails: t.bousema{at}ncmls.ru.nl, t.arens{at}mmb.umcn.nl, and r.sauerwein{at}mmb.umcn.nl. Chris J. Drakeley and Rein Houben, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom, E-mails: chris.drakeley{at}lshtm.ac.uk and rein.houben{at}lshtm.ac.uk. Petra F. Mens and Henk Schallig, KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands, E-mails: p.mens{at}kit.nl and h.schallig{at}kit.nl. Sabah A. Omar, Kenya Medical Research Institute, PO Box 54840, Nairobi, Kenya, E-mail: osabah{at}kemri.org. Louis C. Gouagna, Institut de Recherche pour le Développement, 01 PO Box 182, Ouagadougou, Burkina Faso, E-mail: louis-clement.gouagna{at}ird.bf.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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