Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity.

Quantifying the evolution and impact of antimalarial drug resistance: drug use, spread of resistance, and drug failure over a 12-year period in Papua New Guinea.

BACKGROUND. Antimalarial use is a key factor driving drug resistance and reduced treatment effectiveness in Plasmodium falciparum malaria, but there are few formal, quantitative analyses of this process. METHODS. We analyzed drug usage, drug failure rates, and the frequencies of mutations and haplotypes known to be associated with drug resistance over a 12-year period (1991-2002) in a site in Papua New Guinea. This period included 2 successive treatment policies: amodiaquine (AQ) or chloroquine (CQ) from 1991 through 2000 and their subsequent replacement by sulfadoxine-pyrimethamine (SP) plus AQ or SP plus CQ. RESULTS. Drug use approximated 1 treatment per person-year and was associated with increasing frequencies of pfcrt and pfmdr1 mutations and of treatment failure. The frequency of pfdhfr mutations also increased, especially after the change in treatment policy. Treatment failure rates multiplied by 3.5 between 1996 and 2000 but then decreased dramatically after treatment policy change. CONCLUSIONS. With high levels of resistance to CQ, AQ, and SP, the deployment of the combination of both drugs appears to increase clinical effectiveness but does not decelerate growth of resistance. Our estimates of mutation and haplotype frequencies provide estimates of selection coefficients acting in this environment, which are key parameters for understanding the dynamics of resistance.

Plasmodium falciparum resistance to anti-malarial drugs in Papua New Guinea: evaluation of a community-based approach for the molecular monitoring of resistance.

BACKGROUND: Molecular monitoring of parasite resistance has become an important complementary tool in establishing rational anti-malarial drug policies. Community surveys provide a representative sample of the parasite population and can be carried out more rapidly than accrual of samples from clinical cases, but it is not known whether the frequencies of genetic resistance markers in clinical cases differ from those in the overall population, or whether such community surveys can provide good predictions of treatment failure rates. METHODS: Between 2003 and 2005, in vivo drug efficacy of amodiaquine or chloroquine plus sulphadoxine-pyrimethamine was determined at three sites in Papua New Guinea. The genetic drug resistance profile (i.e., 33 single nucleotide polymorphisms in Plasmodium falciparum crt, mdr1, dhfr, dhps, and ATPase6) was concurrently assessed in 639 community samples collected in the catchment areas of the respective health facilities by using a DNA microarray-based method. Mutant allele and haplotype frequencies were determined and their relationship with treatment failure rates at each site in each year was investigated. RESULTS: PCR-corrected in vivo treatment failure rates were between 12% and 28% and varied by site and year with variable longitudinal trends. In the community samples, the frequencies of mutations in pfcrt and pfmdr1 were high and did not show significant changes over time. Mutant allele frequencies in pfdhfr were moderate and those in pfdhps were low. No mutations were detected in pfATPase6. There was much more variation between sites than temporal, within-site, variation in allele and haplotype frequencies. This variation did not correlate well with treatment failure rates. Allele and haplotype frequencies were very similar in clinical and community samples from the same site. CONCLUSIONS: The relationship between parasite genetics and in vivo treatment failure rate is not straightforward. The frequencies of genetic anti-malarial resistance markers appear to be very similar in community and clinical samples, but cannot be used to make precise predictions of clinical outcome. Thus, indicators based on molecular data have to be considered with caution and interpreted in the local context, especially with regard to prior drug usage and level of pre-existing immunity. Testing community samples for molecular drug resistance markers is a complementary tool that should help decision-making for the best treatment options and appropriate potential alternatives.

Three different Plasmodium species show similar patterns of clinical tolerance of malaria infection.

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.

Plasmodium vivax and mixed infections are associated with severe malaria in children: a prospective cohort study from Papua New Guinea.

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.

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.

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.

Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon.

BACKGROUND: Asymptomatic individuals are one of the major challenges for malaria elimination programs in endemic areas. In the absence of clinical symptoms and with a lower parasite density they constitute silent reservoirs considered important for maintaining transmission of human malaria. Studies from Brazil have shown that infected individuals may carry these parasites for long periods. RESULTS: Patients were selected from three periurban endemic areas of the city of Manaus, in the western Brazilian Amazon. Symptomatic and asymptomatic patients with positive thick blood smear and quantitative real-time PCR (qPCR) positive for Plasmodium vivax were invited to participate in the study. A standardised pvs25 gene amplification by qPCR was used for P. vivax gametocytes detection. Anopheles aquasalis were fed using membrane feeding assays (MFA) containing blood from malaria patients. Parasitemia of 42 symptomatic and 25 asymptomatic individuals was determined by microscopic examination of blood smears and qPCR. Parasitemia density and gametocyte density were assessed as determinants of infection rates and oocysts densities. A strong correlation between gametocyte densities (microscopy and molecular techniques) and mosquito infectivity (P < 0.001) and oocysts median numbers (P < 0.05) was found in both groups. The ability to infect mosquitoes was higher in the symptomatic group (41%), but infectivity in the asymptomatic group was also seen (1.42%). CONCLUSIONS: Although their infectivity to mosquitoes is relatively low, given the high prevalence of P. vivax asymptomatic carriers they are likely to play and important role in malaria transmission in the city of Manaus. The role of asymptomatic infections therefore needs to be considered in future malaria elimination programs in Brazil.

Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites.

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum.

The epidemiology of malaria in Papua New Guinea.

Papua New Guinea (PNG) is a patchwork of different ecological zones, inhabited by human populations of exceptional cultural and linguistic diversity. This results in complex variations in vector ecology and malaria epidemiology. Malaria is the main cause of morbidity in many health facilities in lowland areas, but it is absent in much of the highlands. All four human malaria species occur, but endemicity varies widely, with Plasmodium falciparum locally reaching holo-endemic levels that are rarely found outside sub-Saharan Africa. The high frequency of Plasmodium vivax is an important difference to most African situations. PNG is therefore a prime location for studies of interactions between different parasite species, and of the biology of local human genetic adaptation and its implications for malaria morbidity and mortality.

Random spatial distribution of Schistosoma mansoni and hookworm infections among school children within a single village.

Schistosomes and soil-transmitted helminths currently infect a third of the world's human population. An important feature of these parasitic infections is their focal distribution, which has significant implications for control. Only a few studies have been carried out at the microepidemiological scale, comparing infection levels among individuals or households within a single village. In this study, data are presented from a cross-sectional survey, examining all children attending a primary school in rural Côte d'Ivoire over several consecutive days for Schistosoma mansoni, soil-transmitted helminths, and intestinal protozoa. All houses in the main village were mapped, and school children were linked to these households for small-area spatial analyses. Comparison between the 260 school children who live within the main village and the 89 children who reside in nearby settlements revealed significant differences in the overall prevalence and intensity of infections with S. mansoni and hookworm, confirming the focal nature of these 2 parasites. On the other hand, S. mansoni and hookworm infections exhibited random spatial patterns within the main village. The validity of these results is discussed in the context of this epidemiological setting, drawing attention to the issue of scale. Our findings have direct implications for intervention because they call for a uniform, community-wide approach to control schistosomiasis and soil-transmitted helminthiasis. Implementation can be relatively straightforward, and the proposed control approach might be cost-effective and prove sustainable.

Evaluation of a novel magneto-optical method for the detection of malaria parasites.

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as ∼ 40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/µL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/µL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs.

Critical factors in fatal collisions of adult cyclists with automobiles.

This article evaluates, by means of multivariate regression, critical factors influencing the collisions of motor vehicles with adult (over 17 years) cyclists that result in fatal injury of cyclists. The analysis is based on the database of the Traffic Police of Czech Republic from the time period 1995-2007. The results suggest that the most consequential categories of factors under study are: inappropriate driving speed of automobile; the head-on crash; and night-time traffic in places without streetlights. The cyclists' faults are of most serious consequence on crossroads when cyclists deny the right of way. Males are more likely to suffer a fatal injury due to a collision with a car than females. The most vulnerable age group are cyclists above 65 years. A fatal injury of a cyclist is more often driver's fault than cyclist's (598 vs. 370). In order to reduce the fatal risk, it is recommended to separate the road traffic of motor vehicles from bicyclists in critical road-sections; or, at least, to reduce speed limits there.

Lack of multiple copies of pfmdr1 gene in Papua New Guinea.

We describe here the results of an analysis of Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene copy number from 440 field isolates from Papua New Guinea. No multiple copies of the gene were found, which corresponds to the lack of usage of mefloquine. These data extend regional knowledge about the distribution of multidrug-resistant P. falciparum.

Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1.

BACKGROUND: Molecular markers for sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax have been reported. However, data on the molecular correlates involved in the development of resistance to 4-aminoquinolines and their association with the in vivo treatment response are scarce. METHODS: We assessed pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L) and pvmdr1 (Y976F and F1076L) mutations in 94 patients who received amodiaquine (AQ) plus SP in Papua New Guinea (PNG). We then investigated the association between parasite genotype and treatment response. RESULTS: The treatment failure (TF) rate reached 13%. Polymorphisms in pvdhfr F57L, S58R, T61M, and S117T/N and in pvmdr1 Y976F were detected in 60%, 67%, 20%, 40%, and 39% of the samples, respectively. The single mutant pvdhfr 57 showed the strongest association with TF (odds ratio [OR], 9.04; P= .01). The combined presence of the quadruple mutant pvdhfr 57L+58R+61M+117T and pvmdr1 mutation 976F was the best predictor of TF (OR, 8.56; P= .01). The difference in TF rates between sites was reflected in the genetic drug-resistance profile of the respective parasites. CONCLUSIONS: The present study identified a new molecular marker in pvmdr1 that is associated with the in vivo response to AQ+SP. We suggest suitable marker sets with which to monitor P. vivax resistance against AQ+SP in countries where these drugs are used.