Molecular-based assay for simultaneous detection of four Plasmodium spp. and Wuchereria bancrofti infections.

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)-based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction-fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85-93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.

Mapping the prevalence of malaria in rural Papua New Guinea using a geographic information system.

The application of geographic information systems (GIS) technology to malaria surveillance presents an opportunity for focusing intervention and prevention activities in the areas most affected. We used GIS technology to map the prevalence of malaria in the Wosera Health and Demographic Surveillance Site, East Sepik Province, Papua New Guinea (PNG). Malaria, demographic and GIS data collected between 2001 and 2003 were aggregated and analysed. This was achieved by geo-coding or linking the prevalence and demographic data to the village location. All GIS manipulation and cartographic displays were performed in MapInfo. The results suggest that malaria is endemic with high prevalence as observed across the 3 surveyed years. The optimized implementation of GIS can be of tremendous benefit in the fight against malaria and other public health challenges in PNG.

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.

The impact of repeated rounds of mass drug administration with diethylcarbamazine plus albendazole on bancroftian filariasis in Papua New Guinea.

BACKGROUND: This study employed various monitoring methods to assess the impact of repeated rounds of mass drug administration (MDA) on bancroftian filariasis in Papua New Guinea, which has the largest filariasis problem in the Pacific region. METHODOLOGY/PRINCIPAL FINDINGS: Residents of rural villages near Madang were studied prior to and one year after each of three rounds of MDA with diethylcarbamazine plus albendazole administered per World Health Organization (WHO) guidelines. The mean MDA compliance rate was 72.9%. Three rounds of MDA decreased microfilaremia rates (Mf, 1 ml night blood by filter) from 18.6% pre-MDA to 1.3% after the third MDA (a 94% decrease). Mf clearance rates in infected persons were 71%, 90.7%, and 98.1% after 1, 2, and 3 rounds of MDA. Rates of filarial antigenemia assessed by card test (a marker for adult worm infection) decreased from 47.5% to 17.1% (a 64% decrease) after 3 rounds of MDA. The filarial antibody rate (IgG(4) antibodies to Bm14, an indicator of filarial infection status and/or exposure to mosquito-borne infective larvae) decreased from 59.3% to 25.1% (a 54.6% decrease). Mf, antigen, and antibody rates decreased more rapidly in children <11 years of age (by 100%, 84.2%, and 76.8%, respectively) relative to older individuals, perhaps reflecting their lighter infections and shorter durations of exposure/infection prior to MDA. Incidence rates for microfilaremia, filarial antigenemia, and antifilarial antibodies also decreased significantly after MDA. Filarial DNA rates in Anopheles punctulatus mosquitoes that had recently taken a blood meal decreased from 15.1% to 1.0% (a 92.3% decrease). CONCLUSIONS/SIGNIFICANCE: MDA had dramatic effects on all filariasis parameters in the study area and also reduced incidence rates. Follow-up studies will be needed to determine whether residual infection rates in residents of these villages are sufficient to support sustained transmission by the An. punctulatus vector. Lymphatic filariasis elimination should be feasible in Papua New Guinea if MDA can be effectively delivered to endemic populations.

Mass drug administration trial to eliminate lymphatic filariasis in Papua New Guinea: changes in microfilaremia, filarial antigen, and Bm14 antibody after cessation.

Laboratory tools to monitor infection burden are important to evaluate progress and determine endpoints in programs to eliminate lymphatic filariasis. We evaluated changes in Wuchereria bancrofti microfilaria, filarial antigen and Bm14 antibody in individuals who participated in a five-year mass drug administration trial in Papua New Guinea. Comparing values before treatment and one year after four annual treatments, the proportion of microfilaria positive individuals declined to the greatest degree, with less marked change in antibody and antigen rates. Considering children as sentinel groups who reflect recent transmission intensity, children surveyed before the trial were more frequently microfilaria and antibody positive than those examined one year after the trial stopped. In contrast, antigen positive rates were similar in the two groups. All infection indicators continued to decline five years after cessation of mass drug administration; Bm14 antibody persisted in the greatest proportion of individuals. These data suggest that Bm14 antibody may be a sensitive test to monitor continuing transmission during and after mass drug administration aimed at eliminating transmission of lymphatic filariasis.

Reduced Plasmodium vivax erythrocyte infection in PNG Duffy-negative heterozygotes.

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.

Efficacy of single-dose diethylcarbamazine compared with diethylcarbamazine combined with albendazole against Wuchereria bancrofti infection in Papua New Guinea.

The efficacy of diethylcarbamazine alone was compared with diethylcarbamazine plus albendazole in residents of an island in Papua New Guinea endemic for Wuchereria bancrofti. There was no statistically significant difference between the two drug regimens in decreasing the microfilaria positive rate at 12 and 24 months after a single-dose treatment with either regimen, e.g., 50.0% clearance of microfilaria at 24 months for diethylcarbamazine alone versus 65.7% clearance of microfilaria for diethylcarbamazine plus albendazole (P > 0.05). In contrast, diethylcarbamazine plus albendazole resulted in a significant decrease in Og4C3 antigen prevalence (17%; P = 0.003) at 24 months whereas diethylcarbamazine did not (10%; P = 0.564). These data showed no statistically significant difference in the efficacy of the two drug regimens in lowering the microfilaria reservoir, but they support the use of diethylcarbamazine combined with albendazole in mass treatment programs on the basis of greater activity against adult worms.

Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis.

In Papua New Guinea (PNG), complex patterns of malaria commonly include single and mixed infections of Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Here, we assess recent epidemiologic characteristics of Plasmodium blood-stage infections in the Wosera region through four cross-sectional surveys (August 2001 to June 2003). Whereas previous studies performed here have relied on blood smear/light microscopy (LM) for diagnosing Plasmodium species infections, we introduce a newly developed, post-polymerase chain reaction (PCR), semi-quantitative, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). A direct comparison of the two methods for > 1,100 samples showed that diagnosis was concordant for > 80% of the analyses performed for P. falciparum (PF), P. vivax (PV), and P. malariae (PM). Greater sensitivity of the LDR-FMA accounted for 75% of the discordance between diagnoses. Based on LM, the prevalence of blood-stage PF, PV, and PM infections was found to be markedly reduced compared with an early 1990s survey. In addition, there were significant shifts in age distribution of infections, with PV becoming the most common parasite in children < 4 years of age. Consistent with previous studies, prevalence of all Plasmodium species infections increased significantly in samples analyzed by the PCR-based LDR-FMA. This increase was most pronounced for PM, PO, and mixed infections and in adolescent (10-19 years) and adult age groups, suggesting that LM may lead to under-reported prevalence of less common Plasmodium species, infection complexity, and a skewed distribution of infections towards younger age groups. This study shows that the application of LDR-FMA diagnosis in large epidemiologic studies or malaria control interventions is feasible and may contribute novel insights regarding the epidemiology of malaria.

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein.

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.

Mass treatment to eliminate filariasis in Papua New Guinea.

BACKGROUND: The global initiative to eradicate bancroftian filariasis currently relies on mass treatment with four to six annual doses of antifilarial drugs. The goal is to reduce the reservoir of microfilariae in the blood to a level that is insufficient to maintain transmission by the mosquito vector. METHODS: In nearly 2500 residents of Papua New Guinea, we prospectively assessed the effects of four annual treatments with a single dose of diethylcarbamazine plus ivermectin or diethylcarbamazine alone on the incidence of microfilariae-positive infections, the severity of lymphatic disease, and the rate of transmission of Wuchereria bancrofti by mosquitoes. Random assignment to treatment regimens was carried out according to the village of residence, and villages were categorized as having moderate or high rates of transmission. RESULTS: The four annual treatments with either drug regimen were taken by 77 to 86 percent of the members of the population who were at least five years old; treatments were well tolerated. The proportion with microfilariae-positive infections decreased by 86 to 98 percent, with a greater reduction in areas with a moderate rate of transmission than in those with a high rate. The respective aggregate frequencies of hydrocele and leg lymphedema were 15 percent and 5 percent before the trial began, and 5 percent (P<0.001) and 4 percent (P=0.04) after five years. Hydrocele and leg lymphedema were eliminated in 87 percent and 69 percent, respectively, of those who had these conditions at the outset. The rate of transmission by mosquitoes decreased substantially, and new microfilariae-positive infections in children were almost completely prevented over the five-year study period. CONCLUSIONS: Annual mass treatment with drugs such as diethylcarbamazine can virtually eliminate the reservoir of microfilariae and greatly reduce the frequency of clinical lymphatic abnormalities due to bancroftian filariasis. Eradication may be possible in areas with moderate rates of transmission, but longer periods of treatment or additional control measures may be necessary in areas with high rates of transmission.

Age-dependent cellular immune responses to Plasmodium vivax Duffy binding protein in humans.

The Plasmodium vivax merozoite Duffy binding protein (DBP) contains a cysteine-rich region II (DBPII) that binds to the Duffy Ag receptor for chemokines on erythrocytes, which is essential for parasite invasion. Cellular immune responses to DBPII have not been reported in P. vivax endemic populations, although they may contribute to partial acquired immunity. To examine host cellular immunity to DBPII and identify major T cell epitopes, PBMCs from 107 individuals (2-68 years old) were examined for cytokine production by ELISPOT and/or ELISA to rDBP and overlapping peptides (displaced by 2 aa spanning a 170-aa region of DBPII corresponding to the critical binding motif to the Duffy Ag receptor for chemokines). In P. vivax-exposed subjects, 60 and 71% generated significant rDBP-induced IFN-gamma and IL-10 production, respectively, 11% stimulated IL-2, and IL-5 and IL-13 were not detected. Children <5 years of age had reduced levels and frequency of rDBP-induced IL-10 and IFN-gamma production compared with partially immune older children and adults (p < 0.01). Five major T cell epitopes were identified. Three of these T cell epitopes contained polymorphic residues present in the population. Peptides synthesized corresponding to these variants induced IFN-gamma and IL-10 production to one variant and little response to the other variant in the same individual. These results demonstrate age-dependent and variant-specific cellular immune responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in endemic populations.

Age-acquired immunity to a Plasmodium vivax invasion ligand, the duffy binding protein.

The interaction between the Plasmodium vivax merozoite Duffy binding protein region II (DBPII) and the human erythrocyte Duffy antigen leads to infection. Highly polymorphic regions of this protein may have arisen as a mechanism to avoid host immunity. To examine whether immunity to P. vivax is directed against these polymorphic regions of DBPII, age-associated changes in the frequency of specific DBPII alleles among 358 P. vivax-positive Papua New Guineans were examined. Although the overall number and diversity of DBPII haplotypes simultaneously infecting an individual decreased with increasing age, only certain alleles at particular loci declined in frequency, indicating preferential immune selection against these alleles. One such polymorphic locus formed part of a B cell epitope, and antibodies from exposed individuals differentially recognized alleles at this locus. Therefore, acquisition of strain-specific age-acquired immunity is partially directed against polymorphic motifs within P. vivax DBPII, suggesting that these polymorphisms are maintained and likely arose under immune pressure in the host.