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    Plasmodium vivax and mixed infections are associated with severe malaria in children: A prospective cohort study from Papua New Guinea
    Genton, B ; D'Acremont, V ; Rare, L ; Baea, K ; Reeder, JC ; Alpers, MP ; Mueller, I ; Rogerson, S (PUBLIC LIBRARY SCIENCE, 2008-06-01)
    BACKGROUND: Severe malaria (SM) is classically associated with Plasmodium falciparum infection. Little information is available on the contribution of P. vivax to severe disease. There are some epidemiological indications that P. vivax or mixed infections protect against complications and deaths. A large morbidity surveillance conducted in an area where the four species coexist allowed us to estimate rates of SM among patients infected with one or several species. METHODS AND FINDINGS: This was a prospective cohort study conducted within the framework of the Malaria Vaccine Epidemiology and Evaluation Project. All presumptive malaria cases presenting at two rural health facilities over an 8-y period were investigated with history taking, clinical examination, and laboratory assessment. Case definition of SM was based on the World Health Organization (WHO) criteria adapted for the setting (i.e., clinical diagnosis of malaria associated with asexual blood stage parasitaemia and recent history of fits, or coma, or respiratory distress, or anaemia [haemoglobin < 5 g/dl]). Out of 17,201 presumptive malaria cases, 9,537 (55%) had a confirmed Plasmodium parasitaemia. Among those, 6.2% (95% confidence interval [CI] 5.7%-6.8%) fulfilled the case definition of SM, most of them in children <5 y. In this age group, the proportion of SM was 11.7% (10.4%-13.2%) for P. falciparum, 8.8% (7.1%-10.7%) for P. vivax, and 17.3% (11.7%-24.2%) for mixed P. falciparum and P. vivax infections. P. vivax SM presented more often with respiratory distress than did P. falciparum (60% versus 41%, p = 0.002), but less often with anaemia (19% versus 41%, p = 0.0001). CONCLUSION: P. vivax monoinfections as well as mixed Plasmodium infections are associated with SM. There is no indication that mixed infections protected against SM. Interventions targeted toward P. falciparum only might be insufficient to eliminate the overall malaria burden, and especially severe disease, in areas where P. falciparum and P. vivax coexist.
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    Microsatellite polymorphism within pfcrt provides evidence of continuing evolution of chloroquine-resistant alleles in Papua New Guinea
    DaRe, JT ; Mehlotra, RK ; Michon, P ; Mueller, I ; Reeder, J ; Sharma, YD ; Stoneking, M ; Zimmerman, PA (BMC, 2007-03-21)
    BACKGROUND: Polymorphism in the pfcrt gene underlies Plasmodium falciparum chloroquine resistance (CQR), as sensitive strains consistently carry lysine (K), while CQR strains carry threonine (T) at the codon 76. Previous studies have shown that microsatellite (MS) haplotype variation can be used to study the evolution of CQR polymorphism and to characterize intra- and inter-population dispersal of CQR in Papua New Guinea (PNG). METHODS: Here, following identification of new polymorphic MS in introns 2 and 3 within the pfcrt gene (msint2 and msint3, respectively), locus-by-locus and haplotype heterozygosity (H) analyses were performed to determine the distribution of this intronic polymorphism among pfcrt chloroquine-sensitive and CQR alleles. RESULTS: For MS flanking the pfcrt CQR allele, H ranged from 0.07 (B5M77, -18 kb) to 0.094 (9B12, +2 kb) suggesting that CQ selection pressure was responsible for strong homogenisation of this gene locus. In a survey of 206 pfcrt-SVMNT allele-containing field samples from malaria-endemic regions of PNG, H for msint2 was 0.201. This observation suggests that pfcrt msint2 exhibits a higher level of diversity than what is expected from the analyses of pfcrt flanking MS. Further analyses showed that one of the three haplotypes present in the early 1980's samples has become the predominant haplotype (frequency = 0.901) in CQR parasite populations collected after 1995 from three PNG sites, when CQR had spread throughout malaria-endemic regions of PNG. Apparent localized diversification of pfcrt haplotypes at each site was also observed among samples collected after 1995, where minor CQR-associated haplotypes were found to be unique to each site. CONCLUSION: In this study, a higher level of diversity at MS loci within the pfcrt gene was observed when compared with the level of diversity at pfcrt flanking MS. While pfcrt (K76T) and its immediate flanking region indicate homogenisation in PNG CQR parasite populations, pfcrt intronic MS variation provides evidence that the locus is still evolving. Further studies are needed to determine whether these intronic MS introduce the underlying genetic mechanisms that may generate pfcrt allelic diversity.
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    Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission
    Karl, S ; David, M ; Moore, L ; Grimberg, BT ; Michon, P ; Mueller, I ; Zborowski, M ; Zimmerman, PA (BMC, 2008-04-25)
    BACKGROUND: Aggregated haemozoin crystals within malaria-infected erythrocytes confer susceptibility of parasitized cells to a magnetic field. Here the utility of this method for diagnosis of human malaria is evaluated in a malaria-endemic region of Papua New Guinea (PNG). METHODS AND FINDINGS: Individuals with Plasmodium falciparum malaria symptoms (n = 55) provided samples for conventional blood smear (CBS) and magnetic deposition microscopy (MDM) diagnosis. Standard Giemsa staining and light microscopy was performed to evaluate all preparations. Plasmodium falciparum parasitaemia observed on MDM slides was consistently higher than parasitaemia observed by (CBS) for ring (CBS = 2.6 vs. MDM = 3.4%; t-test P-value = 0.13), trophozoite (CBS = 0.5 vs. MDM = 1.6%; t-test P-value = 0.01), schizont (CBS = 0.003 vs. MDM = 0.1%; t-test P-value = 0.08) and gametocyte (CBS = 0.001 vs. MDM = 0.4%; t-test P-value = 0.0002) parasitaemias. Gametocyte prevalence determined by CBS compared to MDM increased from 7.3% to 45%, respectively. CONCLUSION: MDM increased detection sensitivity of P. falciparum-infected, haemozoin-containing erythrocytes from infected humans while maintaining detection of ring-stage parasites. Gametocyte prevalence five-fold higher than observed by CBS suggests higher malaria transmission potential in PNG endemic sites compared to previous estimates.
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    Heterogeneous distribution of Plasmodium falciparum drug resistance haplotypes in subsets of the host population
    Schoepflin, S ; Marfurt, J ; Goroti, M ; Baisor, M ; Mueller, I ; Felger, I (BMC, 2008-05-06)
    BACKGROUND: The emergence of drug resistance is a major problem in malaria control. For mathematical modelling of the transmission and spread of drug resistance the determinant parameters need to be identified and measured. The underlying hypothesis is that mutations associated with drug resistance incur fitness costs to the parasite in absence of drug pressure. The distribution of drug resistance haplotypes in different subsets of the host population was investigated. In particular newly acquired haplotypes after radical cure were characterized and compared to haplotypes from persistent infections. METHODS: Mutations associated with antimalarial drug resistance were analysed in parasites from children, adults, and new infections occurring after treatment. Twenty-five known single nucleotide polymorphisms from four Plasmodium falciparum genes associated with drug resistance were genotyped by DNA chip technology. RESULTS: Haplotypes were found to differ between subsets of the host population. A seven-fold mutated haplotype was significantly reduced in adults compared to children and new infections, whereas parasites harbouring fewer mutations were more frequent in adults. CONCLUSION: The reduced frequency of highly mutated parasites in chronic infections in adults is likely a result of fitness costs of drug resistance that increases with number of mutations and is responsible for reduced survival of mutant parasites.
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    The usefulness of twenty-four molecular markers in predicting treatment outcome with combination therapy of amodiaquine plus sulphadoxine-pyrimethamine against falciparum malaria in Papua New Guinea
    Marfurt, J ; Mueller, I ; Sie, A ; Oa, O ; Reeder, JC ; Smith, TA ; Beck, H-P ; Genton, B (BMC, 2008-04-19)
    BACKGROUND: In Papua New Guinea (PNG), combination therapy with amodiaquine (AQ) or chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) was introduced as first-line treatment against uncomplicated malaria in 2000. METHODS: We assessed in vivo treatment failure rates with AQ+SP in two different areas in PNG and twenty-four molecular drug resistance markers of Plasmodium falciparum were characterized in pre-treatment samples. The aim of the study was to investigate the association between infecting genotype and treatment response in order to identify useful predictors of treatment failure with AQ+SP. RESULTS: In 2004, Day-28 treatment failure rates for AQ+SP were 29% in the Karimui and 19% in the South Wosera area, respectively. The strongest independent predictors for treatment failure with AQ+SP were pfmdr1 N86Y (OR = 7.87, p < 0.01) and pfdhps A437G (OR = 3.44, p < 0.01). Mutations found in CQ/AQ related markers pfcrt K76T, A220S, N326D, and I356L did not help to increase the predictive value, the most likely reason being that these mutations reached almost fixed levels. Though mutations in SP related markers pfdhfr S108N and C59R were not associated with treatment failure, they increased the predictive value of pfdhps A437G. The difference in treatment failure rate in the two sites was reflected in the corresponding genetic profile of the parasite populations, with significant differences seen in the allele frequencies of mutant pfmdr1 N86Y, pfmdr1 Y184F, pfcrt A220S, and pfdhps A437G. CONCLUSION: The study provides evidence for high levels of resistance to the combination regimen of AQ+SP in PNG and indicates which of the many molecular markers analysed are useful for the monitoring of parasite resistance to combinations with AQ+SP.
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    Reduced Plasmodium vivax Erythrocyte Infection in PNG Duffy-Negative Heterozygotes
    Kasehagen, LJ ; Mueller, I ; Kiniboro, B ; Bockarie, MJ ; Reeder, JC ; Kazura, JW ; Kastens, W ; McNamara, DT ; King, CH ; Whalen, CC ; Zimmerman, PA ; Awadalla, P (PUBLIC LIBRARY SCIENCE, 2007-03-28)
    BACKGROUND: Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/-). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. METHODS AND FINDINGS: We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/-) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/- vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. CONCLUSIONS: Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely.
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    Comparison of Plasmodium falciparum allelic frequency distribution in different endemic settings by high-resolution genotyping
    Schoepflin, S ; Valsangiacomo, F ; Lin, E ; Kiniboro, B ; Mueller, I ; Felger, I (BMC, 2009-10-30)
    BACKGROUND: The diversity of genotyping markers of Plasmodium falciparum depends on transmission intensity. It has been reported that the diversity of the merozoite surface protein 2 (msp2) is greater in areas of high compared to low endemicity, however, results for msp1 were inconsistent. These previous reports relied on low resolution genotyping techniques. METHODS: In the present study, a high-resolution capillary electrophoresis-based technique was applied to genotype samples from areas of different endemicity in Papua New Guinea and Tanzania. For both endemic settings, the diversity of msp1 and msp2 was investigated; the mean multiplicity of infection (MOI) and the FST values were determined to investigate whether more accurate sizing generates different results. RESULTS AND CONCLUSION: The results of the present study confirmed previous reports of a higher mean MOI for both marker genes and increased genetic diversity in areas of higher endemicity as estimated by the total number of distinct alleles for msp2. For msp1 a minor increase in diversity was observed. Measures of between population variance in allele frequencies (FST) indicated little genetic differentiation for both marker genes between the two populations from different endemic settings. MOI adjusted for the probability of multiple infections sharing the same allele was estimated by using the msp2 allele frequency distribution and the distribution of observed numbers of concurrent infections. For the high-resolution typing technique applied in this study, this adjustment made little difference to the estimated mean MOI compared to the observed mean MOI.
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    High sensitivity detection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea.
    Mueller, I ; Widmer, S ; Michel, D ; Maraga, S ; McNamara, DT ; Kiniboro, B ; Sie, A ; Smith, TA ; Zimmerman, PA (Springer Science and Business Media LLC, 2009-03-11)
    BACKGROUND: When diagnosed by standard light microscopy (LM), malaria prevalence can vary significantly between sites, even at local scale, and mixed species infections are consistently less common than expect in areas co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. The development of a high-throughput molecular species diagnostic assay now enables routine PCR-based surveillance of malaria infections in large field and intervention studies, and improves resolution of species distribution within and between communities. METHODS: This study reports differences in the prevalence of infections with all four human malarial species and of mixed infections as diagnosed by LM and post-PCR ligase detection reaction-fluorescent microsphere (LDR-FMA) assay in 15 villages in the central Sepik area of Papua New Guinea. RESULTS: Significantly higher rates of infection by P. falciparum, P. vivax, P. malariae and Plasmodium ovale were observed in LDR-FMA compared to LM diagnosis (p < 0.001). Increases were particularly pronounced for P. malariae (3.9% vs 13.4%) and P. ovale (0.0% vs 4.8%). In contrast to LM diagnosis, which suggested a significant deficit of mixed species infections, a significant excess of mixed infections over expectation was detected by LDR-FMA (p < 0.001). Age of peak prevalence shifted to older age groups in LDR-FMA diagnosed infections for P. falciparum (LM: 7-9 yrs 47.5%, LDR-FMA: 10-19 yrs 74.2%) and P. vivax (LM: 4-6 yrs 24.2%, LDR-FMA: 7-9 yrs 50.9%) but not P. malariae infections (10-19 yrs, LM: 7.7% LDR-FMA: 21.6%). Significant geographical variation in prevalence was found for all species (except for LM-diagnosed P. falciparum), with the extent of this variation greater in LDR-FMA than LM diagnosed infections (overall, 84.4% vs. 37.6%). Insecticide-treated bednet (ITN) coverage was also the dominant factor linked to geographical differences in Plasmodium species infection prevalence explaining between 60.6% - 74.5% of this variation for LDR-FMA and 81.8% - 90.0% for LM (except P. falciparum), respectively. CONCLUSION: The present study demonstrates that application of molecular diagnosis reveals patterns of malaria risk that are significantly different from those obtained by standard LM. Results provide insight relevant to design of malaria control and eradication strategies.
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    Malaria - a major health problem within an oil palm plantation around Popondetta, Papua New Guinea
    Pluess, B ; Mueller, I ; Levi, D ; King, G ; Smith, TA ; Lengeler, C (BIOMED CENTRAL LTD, 2009-04-08)
    BACKGROUND: For companies operating in malaria endemic countries, malaria represents a substantial risk to workers and their dependants, and can lead to significantly reduced worker productivity. This study provides an overview of the malaria epidemiology within an oil palm plantation in Popondetta, south-eastern Papua New Guinea, its implication for the company with its employees and their families and the potential for control. METHODS: In 2006, we carried out a cross-sectional study within six company villages, which included the determination of parasite rates by conventional microscopy, interviews and haemoglobin measurements. Passive surveillance data were collected from the 13 company aid posts for the years 2005 and 2006. RESULTS: Malaria prevalence was found to be high: all-age prevalence was 33.5% (95% CI 30.1-37.0) in 723 individuals. Plasmodium falciparum was the dominant species, followed by Plasmodium vivax and Plasmodium malariae. Children between five and nine years of age were most affected (40.3%, 95% CI 0.32-0.49). Haemoglobin levels were found to be low; 11.0 g/dl (95% CI 10.8-11.1) for men and 10.4 g/dl (95% CI 10.3-10.5) for women, respectively. Plasmodium falciparum infections were significantly associated with anaemia (Hb < 10 g/dl). At the aid posts, all malaria cases in 2005 and January-March 2006 were diagnosed by symptoms only, while from April 2006 onwards most cases were tested by rapid diagnostic tests. Between 2005 and 2006, 22,023 malaria cases were diagnosed at the aid posts and malaria accounted for 30-40% of all clinical cases. Of the malaria cases, 13-20% were HOP employees. On average, an employee sick with malaria was absent for 1.8 days, resulting in a total of 9,313 workdays lost between 2005 and 2006. Sleeping outside of the house did not increase the risk of a malaria infection, neither did getting up before 7 am. CONCLUSION: Malaria was found to be a major health burden in the Higaturu Oil Palm plantation, posing a high risk for company staff and their relatives, including expatriates and other non-immune workers. Reducing the malaria risk is a highly recommended investment for the company.
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    Three different Plasmodium species show similar patterns of clinical tolerance of malaria infection
    Mueller, I ; Genton, B ; Rare, L ; Kiniboro, B ; Kastens, W ; Zimmerman, P ; Kazura, J ; Alpers, M ; Smith, TA (BMC, 2009-07-14)
    BACKGROUND: In areas where malaria endemicity is high, many people harbour blood stage parasites without acute febrile illness, complicating the estimation of disease burden from infection data. For Plasmodium falciparum the density of parasitaemia that can be tolerated is low in the youngest children, but reaches a maximum in the age groups at highest risk of infection. There is little data on the age dependence of tolerance in other species of human malaria. METHODS: Parasite densities measured in 24,386 presumptive malaria cases at two local health centres in the Wosera area of Papua New Guinea were compared with the distributions of parasite densities recorded in community surveys in the same area. We then analyse the proportions of cases attributable to each of Plasmodium falciparum, P. vivax, and P. malariae as functions of parasite density and age using a latent class model. These attributable fractions are then used to compute the incidence of attributable disease. RESULTS: Overall 33.3%, 6.1%, and 0.1% of the presumptive cases were attributable to P. falciparum, P. vivax, and P. malariae respectively. The incidence of attributable disease and parasite density broadly follow similar age patterns. The logarithm of the incidence of acute illness is approximately proportion to the logarithm of the parasite density for all three malaria species, with little age variation in the relationship for P. vivax or P. malariae. P. falciparum shows more age variation in disease incidence at given levels of parasitaemia than the other species. CONCLUSION: The similarities between Plasmodium species in the relationships between parasite density and risk of attributable disease are compatible with the hypothesis that pan-specific mechanisms may regulate tolerance to different human Plasmodia. A straightforward mathematical expression might be used to project disease burden from parasite density distributions assessed in community-based parasitological surveys.