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 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.

Parasitological and clinical efficacy of standard treatment regimens against Plasmodium falciparum, P. vivax and P. malariae in Papua New Guinea.

Resistance of Plasmodium falciparum (Pf) and P. vivax (Pv) to standard antimalarials is widespread in Papua New Guinea (PNG). The objective of the study was to assess the rate of clinical treatment failure (TF) and parasite resistance to amodiaquine (AQ), chloroquine (CQ) and quinine+sulfadoxine/pyrimethamine (Q+SP) for malaria in a rural health centre of the East Sepik Province. 179 patients presenting with symptoms and signs of malaria and with Pf (144 patients), Pv (18 patients), P. malariae (Pm) (7 patients) or mixed infection (10 patients) were included. 86 were treated with AQ, 88 with CQ and 5 with Q+SP. 21/179 patients (12%) were not cured or had a recrudescence of symptoms associated with parasitaemia in the 28 days following treatment, 14% after AQ, 10% after CQ and 0% after Q+SP. The proportion of TF was higher (17%) when the analysis population included only the 108 subjects who had a complete follow-up, especially for failure with Pf following AQ treatment (26%). During the 28 days of follow-up, RII or RIII level of resistance in Pf was detected in 55% of the subjects treated with amodiaquine, 30% of those treated with chloroquine and 0% of those treated with quinine+SP. Of the Pv or Pm parasites only one Pv was found to be RII resistant to CQ in the 28-day test. In vitro resistance of Pf to CQ was higher than to AQ (50% versus 27% of 36 parasite samples that grew successfully). The level of TF and parasitological resistance to standard antimalarial drugs was lower in this area than in urban settings, where drugs are more easily available. AQ performed less well than CQ but the difference is likely to be due to the age of the users, ie, their level of immunity, AQ being the first-line drug for young children. These results provided support for the recent change in the policy for the standard treatment of uncomplicated malaria in PNG from AQ or CQ to the combination of AQ+SP or CQ+SP, a recommendation aimed at slowing down the spread of multidrug resistance.

Analysis of Plasmodium falciparum var genes expressed in children from Papua New Guinea.

BACKGROUND: The variable antigen P. falciparum erythrocyte membrane protein-1 (PfEMP1) is a major virulence factor in malaria. A large number of var genes encode PfEMP1, and we hypothesized that a restricted PfEMP1 repertoire determines clinical disease presentation. We conducted a case-control study in Papua New Guinea and analyzed transcribed var genes in naturally infected children. METHODS: var messenger RNA was isolated from 78 children with asymptomatic, mild, or severe malaria. We prepared complementary DNA from the upstream region into the DBL1alpha domain and picked, on average, 20 clones for sequencing. RESULTS: Twenty-five percent of centrally located var genes were shared between children, whereas only 5% of subtelomeric genes were shared, indicating lower diversity in the former group. Linkage between group B or C var upstream sequences and DBL1alpha groups was not observed, which impeded prediction by DBL1alpha analysis. A higher proportion of var group A sequences was detected in symptomatic malaria, and a subgroup of frequently encountered var genes with complex head structure seems to be associated with severe malaria. A subset of var group C genes was frequently expressed in older children with asymptomatic high levels of parasitemia. CONCLUSION: Despite this vast diversity, restricted disease-associated var genes were identified and might be used for innovative interventions based on PfEMP1.

Virulence of malaria is associated with differential expression of Plasmodium falciparum var gene subgroups in a case-control study.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a major pathogenicity factor in falciparum malaria that mediates cytoadherence. PfEMP1 is encoded by approximately 60 var genes per haploid genome. Most var genes are grouped into 3 subgroups: A, B, and C. Evidence is emerging that the specific expression of these subgroups has clinical significance. Using field samples from children from Papua New Guinea with severe, mild, and asymptomatic malaria, we compared proportions of transcripts of var groups, as determined by quantitative polymerase chain reaction. We found a significantly higher proportion of var group B transcripts in children with clinical malaria (mild and severe), whereas a large proportion of var group C transcripts was found in asymptomatic children. These data from naturally infected children clearly show that major differences exist in var gene expression between parasites causing clinical disease and those causing asymptomatic infections. Furthermore, parasites forming rosettes showed a significant up-regulation of var group A transcripts.

Safety and immunogenicity of a three-component blood-stage malaria vaccine (MSP1, MSP2, RESA) against Plasmodium falciparum in Papua New Guinean children.

Combination B is a malaria vaccine that comprises recombinant Plasmodium falciparum (P. falciparum) blood-stage proteins MSP1, MSP2 and RESA, formulated with the adjuvant Montanide ISA 720. A phase I-IIb double-blind randomised placebo-controlled trial was undertaken in 120 children aged 5-9 years. Subjects were randomised in four groups: (i) No sulphadoxine-pyrimethamine (SP)+vaccine, (ii) No SP+placebo, (iii) SP+vaccine, (iv) SP+placebo. 15 microg of each protein were given in the thigh, at both first and second injection (4 weeks apart). The placebo was adjuvant emulsified with saline. No serious or severe AEs occurred. Moderate AEs were seen in 3% of the vaccine and 3% of the placebo recipients after first injection and in 12 and 10% after second injection. The vaccine induced significant antibody responses to all three antigens but triggered an IFN-gamma response to MSP1 only. At Week 12, the IFN-gamma response to MSP1 was substantially higher in the vaccine group where No SP had been given. Combination B proved to be safe and immunogenic in children aged 5-9 years. Vaccine immunogenicity was neither impaired by circulating parasites nor increased after pre-treatment with SP and pre-treatment is not advisable in future trials of malaria vaccines, at least for those including blood-stage antigens.