Antibody responses to Plasmodium falciparum gametocyte antigens during and after malaria attacks in schoolchildren from Madang, Papua New Guinea

Sera from 49 school children in Madang, Papua New Guinea with malaria and follow-up sera from 40 of these cases were tested by competitive ELISA for antibodies capable of inhibiting binding of eight monoclonal antibodies (MoAbs) to Plasmodium falciparum gametocytes. The proportion of sera inhibiting each MoAb ranged from 31.2% to 85.7%. At follow-up, the proportion of inhibitory sera decreased for 3 MoAbs, did not change significantly for 4 MoAbs and increased for one MoAb. When sera were grouped according to whether the follow-up blood slide was positive or negative, further trends emerged for MoAbs against the gamete surface antigen Pfs 48/45. Antibody levels to the IA3-B8 epitope decreased in follow-up positive cases, but remained unchanged for follow-up negative cases. The converse was observed for the IIC5-B10 epitope with an increase of antibody in follow-up positive cases and no change in the negative cases. Amount of antibody to the 3G12/58 epitope decreased when the follow-up was negative but not when it was positive. Increase in antibody to the 3E12/58 epitope occurred at the follow-up sample irrespective of the blood slide result. Thus four distinct patterns of longitudinal antibody response were observed against four epitopes on the same molecule.     

In vitro sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea

Objective Recent clinical studies have shown high rates of malaria treatment failure in endemic areas of Papua New Guinea (PNG), necessitating a change of treatment from chloroquine (CQ) or amodiaquine (AQ) plus sulphadoxine‐pyrimethamine to the artemisinin combination therapy (ACT) artemether plus lumefantrine (LM). To facilitate the monitoring of antimalarial drug resistance in this setting, we assessed the in vitro sensitivity of Plasmodium falciparum isolates from Madang Province. Methods A validated colorimetric lactate dehydrogenase assay was used to assess growth inhibition of 64 P. falciparum isolates in the presence of nine conventional or novel antimalarial drugs [CQ, AQ, monodesethyl‐amodiaquine (DAQ), piperaquine (PQ), naphthoquine (NQ), mefloquine (MQ), LM, dihydroartemisinin and azithromycin (AZ)]. Results The geometric mean (95% confidence interval) concentration required to inhibit parasite growth by 50% (IC₅₀) was 167 (141–197) nm for CQ, and 82% of strains were resistant (threshold 100 nm ), consistent with near‐fixation of the CQ resistance‐associated pfcrt allele in PNG. Except for AZ [8.351 (5.418–12.871) nm ], the geometric mean IC₅₀ for the other drugs was <20 nm . There were strong associations between the IC₅₀s of 4‐aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ) and aryl aminoalcohol (MQ) compounds suggesting cross‐resistance, but LM IC₅₀ only correlated with that of MQ. Conclusions Most PNG isolates are resistant to CQ in vitro but not to other ACT partner drugs. The non‐isotopic semi‐automated high‐throughput nature of the Plasmodium lactate dehydrogenase assay facilitates the convenient serial assessment of local parasite sensitivity, so that emerging resistance can be identified with relative confidence at an early stage.     

Immunoprecipitation of biosynthetically-labelled proteins from different Papua New Guinea Plasmodium falciparum isolates by sera from individuals in the endemic area

The human serum antibody response to Plasmodium falciparum infection in Papua New Guinea has been studied by electrophoretic analysis of immunoprecipitated biosynthetically-labelled malaria proteins from three different isolates maintained in long-term in vitro culture. Differences in protein antigenic composition in different lines have been described and simplified by examination of antigens recognized only by hyperimmune serum. An in vitro assay has been used to screen various human sera containing antimalarial antibody for their ability to inhibit parasite growth and the immunoprecipitation profiles of non-inhibitory sera have been compared with those of a hyperimmune serum pool. In the discussion, emphasis is placed on the value of immunoprecipitation analyses using clinically-defined sera with known in vitro function in the identification of antigens which may be responsible for the induction of host-protective immunity.     

Type 2 (non-insulin-dependent) diabetes mellitus and HLA antigens in Papua New Guinea

Summary HLA phenotypes were studied in 57 Type 2 (non-insulin-dependent) diabetic Papuan patients and the results compared with control subjects of the same Austronesian origin. An association was found between disease and BW62, a split antigen of B15, with corrected probability significant at the 5% level. The frequencies of B13 and BW22 were also increased in diabetic patients but the differences were not statistically significant. Although it has been postulated that Melanesian communities have protection against diabetes, they have a high frequency of BW62, which would imply, from the association found in this study, that susceptibility to Type 2 diabetes has yet to become manifest in them.     

Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies, on gametes of different isolates of Plasmodium falciparum

We have studied the properties of epitopes on Plasmodium falciparum gamete surface protein Pfs 48/45, a target antigen of malaria transmission blocking antibodies. Using a two site immunoradiometric assay we have defined three spacially separate, non-repeated, epitope regions on the peptides representing this antigen. Epitope region I is a target of monoclonal antibodies (MoAbs) which strongly suppress infectivity of gametocytes of P. falciparum to mosquitoes; the effect is complement independent and is mediated as effectively by the monovalent Fab fragments as by intact MoAb. Epitope region II consists of two spacially close subregions, IIa and IIb; variant forms of epitopes IIa and IIb occurred in different isolates of P. falciparum. Epitope region III also showed slight structural modification between isolates. MoAbs against regions II or III were relatively ineffective in suppressing gametocyte infectivity compared to MoAbs against region I. However, certain combinations of MoAbs against regions II and III together acted synergistically to suppress infectivity to mosquitoes. All these epitopes failed to react with MoAb when the antigen was presented in reduced form. A fourth epitope, however, was identified which reacted strongly with MoAb when the antigen was presented in reduced form. The MoAb against this epitope had no effect on the infectivity of gametocytes of P. falciparum to mosquitoes.     

Plasmodium falciparum malaria transmission-blocking immunity under conditions of low endemicity as in Sri Lanka

Sera from acute primary Plasmodium falciparum patients in Sri Lanka were tested for the presence of antibodies against gamete antigens and for their functional effects of transmission blocking activity. Comparisons were made with corresponding data from a previous study from sera of patients from Papua New Guinea where malaria is more highly endemic. Although the prevalence of anti-gamate antibodies in the two groups were broadly similar, the prevalence of infectivity suppressive effects in the Sri Lankan sera (56%) was less than in Papua New Guinea sera (75%), suggesting that the generation of functionally effective transmission blocking antibodies requires prolonged exposure to multiple inoculations of malaria. In Papua New Guinea sera there was a good correlation between transmission blocking effects and antibody responses to Pfs 230, a known target of transmission blocking antibodies. Among the Sri Lankan sera no strong correlation was found between transmission blocking effects and the presence of antibodies to gamete surface antigens Pfs 230 nor Pfs 48/45 as detected by immunoprecipitation of radio-iodinated gamate proteins; a strong correlation was however, found between the intensity of response to gamete surface antigens by IFA and transmission blocking effects of these sera. It is possible therefore, that the antigens identified by IFA include non-protein moieties and that these may be the targets of transmission blocking antibodies in sera from acute primary infections of P. falciparum.     

Epidemiology of malaria in the Papua New Guinean highlands

Objectives To conduct an in‐depth investigation of the epidemiology of malaria in the Papua New Guinea (PNG) highlands and provide a basis for evidence‐based planning and monitoring of intensified malaria control activities. Methods Between December 2000 and July 2005, 153 household‐based, rapid malaria population surveys were conducted in 112 villages throughout the central PNG highlands. The presence of malaria infections was determined by light microscopy and risk factors assessed using a structured questionnaire. The combined dataset from all individually published surveys was reanalysed. Results The prevalence of malaria infections in the different surveys ranged from 0.0% to 41.8% (median 4.3%) in non‐epidemic surveys and 6.6% to 63.2% (median 21.2%, P < 0.001) during epidemics. Plasmodium falciparum was the predominant infection below 1400 m and during epidemics, Plasmodium vivax at altitudes >1600 m. Outside epidemics, prevalence decreased significantly with altitude, was reduced in people using bed nets [odds ratio (OR) = 0.8, P < 0.001] but increased in those sleeping in garden houses (OR = 1.34, P < 0.001) and travelling to highly endemic lowlands (OR = 1.80, P < 0.001). Below 1400 m, malaria was a significant source of febrile illness. At higher altitudes, malaria was only a significant source of febrile illness during epidemic outbreaks, but asymptomatic malaria infections were common in non‐epidemic times. Conclusions Malaria is once again endemic throughout the PNG highlands in areas below 1400–1500 m of altitude with a significant risk of seasonal malaria outbreaks in most area between 1400–1650 m. Ongoing control efforts are likely to result in a substantial reduction in malaria transmission and may even result in local elimination of malaria in higher lying areas.     

Historical epidemics of scrub typhus in Queensland and Papua New Guinea

Undifferentiated febrile diseases (e.g., Mossman fever) from northern Queensland were eventually partially attributed to mite-transmitted rickettsial infections known as scrub typhus or tsutsugamushi fever. Scrub typhus became a major medical threat to military operations in Papua New Guinea during the Second World War and killed more Australian soldiers than malaria in the pre-antibiotic era. Further investigations showed scrub typhus to be an occupational disease of rural workers in north Queensland especially around Cairns and Innisfail. Occasional small epidemics of scrub typhus still occur during military exercises in Queensland, but as scrub typhus is not a reportable disease, its presence in the civilian community is largely unknown. Increased use of serological testing in patients with fever and rash illnesses after exposure in northern Queensland is likely to show that scrub typhus is a modern infection that remains treatable with antibiotics once it is identified.     

Human impacts and Anthropocene environmental change at Lake Kutubu,a Ramsar wetland in Papua New Guinea

The impacts of human-induced environmental change that characterize the Anthropocene are not felt equally across the globe. In the tropics, the potential for the sudden collapse of ecosystems in response to multiple interacting pressures has been of increasing concern in ecological and conservation research. The tropical ecosystems of Papua New Guinea are areas of diverse rainforest flora and fauna, inhabited by human populations that are equally diverse, both culturally and linguistically. These people and the ecosystems they rely on are being put under increasing pressure from mineral resource extraction, population growth, land clearing, invasive species, and novel pollutants. This study details the last ∼90 y of impacts on ecosystem dynamics in one of the most biologically diverse, yet poorly understood, tropical wetland ecosystems of the region. The lake is listed as a Ramsar wetland of international importance, yet, since initial European contact in the 1930s and the opening of mineral resource extraction facilities in the 1990s, there has been a dramatic increase in deforestation and an influx of people to the area. Using multiproxy paleoenvironmental records from lake sediments, we show how these anthropogenic impacts have transformed Lake Kutubu. The recent collapse of algal communities represents an ecological tipping point that is likely to have ongoing repercussions for this important wetland’s ecosystems. We argue that the incorporation of an adequate historical perspective into models for wetland management and conservation is critical in understanding how to mitigate the impacts of ecological catastrophes such as biodiversity loss.

Wetlands provide a range of benefits to human, animal, and plant life and yet are under increasing threat from industrialization, expanding human populations, and climate change. In 1975, the Ramsar Convention was established to promote the sustainable use of wetlands and halt their decline worldwide (1). One of the commitments that parties to the convention make is to declare as early as possible any changes to the ecological character of Ramsar wetlands as a result of developments, pollution, or other human interference (2). This requires knowledge of the “natural” or “baseline” state of the wetland prior to major human interference (3, 4), as well as ongoing monitoring of the site. In some cases, major alterations to wetlands have occurred prior to Ramsar Convention listing, and so the baseline state is not known. Baseline information can be obtained from a multiproxy analysis of sediment core records that built up prior to the altering event (5). These sediment records can also be used as a cost-effective way to track changes in the ecological health of the site during and after such disturbances (3, 4). Here, we use the case study of Lake Kutubu, a Ramsar site in Papua New Guinea (PNG; Fig. 1), as a demonstration of this approach.Open in a separate windowFig. 1.Sediment cores, resource extraction infrastructure, and settlements at Lake Kutubu, PNG. The green dotted line is the catchment boundary. The shaded areas are 1,000 m contours after Bayly et al. (16). This map was created using ArcGIS software by Esri (http://www.esri.com) and contains information from Humanitarian OpenStreetMap Team Open Street Map (HOTOSM) PNG Waterways (https://data.amerigeoss.org/tl/dataset/hotosm_png_waterways) and PNG roads 2014 (https://png-data.sprep.org/dataset/png-roads/resource/2ea995e6-6483-42db-a71a-ad58f9fbb2de), which is made available under the Open Database License (https://opendatacommons.org/licenses/odbl/1-0/).Paleoenvironmental ecological baselines have been successfully developed to define “limits of acceptable change” for Ramsar wetlands in several countries (e.g., refs. 5, 6). The feasibility, cost-effectiveness, and long-term assessment of these baselines allow for any country, independent of economic status, to assess the impacts of resource extraction, other human activities, and climate change on wetland areas. Baselines have the potential to function as ecological tools for environmental justice and to support local communities in tracking the state of their environment. They can also support the Ramsar Convention by providing evidence for ecological impacts and highlighting areas in need of better management.The Ramsar Convention was adopted in PNG on 16 July 1993, and Lake Kutubu (Ramsar site no. 961) was declared a Ramsar site in 1998 (7), due primarily to its exceptional levels of fish endemism supported by food chains based on unique species of autochthonous primary producers (8). It was determined that significant degradation or loss of the lake would substantially reduce the ecological diversity of the region (8). Yet, at the time of listing, the Lake Kutubu region had already been subject to altered scales of human disturbance with the establishment of European administrative bases in the 1930s (9), low-level development from the 1940s to 1970s, and then the much more significant impacts relating to the establishment of petroleum and natural gas projects from the 1980s (10, 11), followed by large-scale land clearance, construction, and immigration into the area (10, 12, 13). A baseline study of the lake’s condition prior to these disturbances is needed to understand the scale of change and to provide a restoration target (14). Such targets are essential where ongoing development pressures and environmental governance issues can otherwise lead to long-term degradation of environments and livelihoods. We apply a paleoenvironmental multiproxy approach to assess the history of contamination, ecological impacts, and deforestation in sediments of Lake Kutubu. The identification of detrimental impacts in Lake Kutubu is crucial to provide guidance for the implementation of mitigation measures in tropical lakes under similar modern pollution pressures. This approach includes the analysis of fire and pollution indicators, aquatic algae, chlorophyll-a, and geochemistry in the lake sediments from before and after resource extraction activities commenced, providing a detailed profile of chemical and ecological changes. These data are compared to historic records and observations on the state of the lake prior to and since the start of resource extraction in the 1990s.     

Immune response to measles vaccine in 6 month old infants in Papua New Guinea

Objective  To assess the efficacy of the current measles immunization schedule in Papua New Guinea, which is to give the first dose at 6 months of age and the second at 9 months.
Methods  Humoral immune response study of 140 Papua New Guinean infants at 6 months of age, measuring measles IgG antibodies by enzyme immunoassay before and 85 days after the 6-month dose of measles vaccine.
Results  After vaccination at 6 months, 35.7% of infants developed a level of measles antibodies consistent with protection (IgG >330 IU/ml); 17.7% had an antibody response (150–330 IU/ml) that is likely to afford some protection; 46.8% had no detectable antibody response (IgG <150 IU/ml). Among 53 infants with no antibody response, 37 (69.5%) developed an antibody response, while 42.4% (37/87) of those with maternal antibodies sero-converted ( P  = 0.002).
Conclusions  Antibody response to measles vaccine was lower than expected at 6 months. While the presence of maternally derived antibodies accounted for some of the limited seroconversion in young infants, other factors are involved. Issues to be considered in determining the value of the first dose of measles vaccination in mid infancy in poor countries are complex and antibody responses are only one factor. Others, such as cell mediated immune responses, the non-specific protective effect of measles vaccine in preventing illness and death and the practicalities of uptake of vaccines at different ages, are also important.     

Human antibody responses to Pfs 230, a sexual stage-specific surface antigen of Plasmodium falciparum: non-responsiveness is a stable phenotype but does not appear to be genetically regulated

The 230 kD gamete surface protein of the malaria parasite Plasmodium falciparum (Pfs 230) is a target of transmission blocking antibodies. Anti-Pfs 230 antibodies are induced following natural infection with malaria but are not found in all P. falciparum-exposed individuals. In this study we have shown that approximately 40% of malaria-exposed Gambians do not make antibodies to the native Pfs 230 molecule. This phenotype is remarkably stable over time and does not appear to be related to age, malaria exposure or major histocompatibility complex genotype. Comparison of antibody responses in twins indicates that the anti-Pfs 230 response is not strictly genetically controlled, but a high degree of concordance within both dizygous and monozygous twin pairs suggests that factors associated with exposure to malaria in childhood may be important in determining the subsequent immune response.     

Human immune recognition of recombinant proteins representing discrete domains of the Plasmodium falciparum gamete surface protein, Pfs230

The 230 kD gamelocyte/gamete-specific surface protein of Plasmodium falciparum, Pfs230, is a target of antibodies which inhibit the development of the parasite inside the mosquito vector. A transmission blocking vaccine based on Pfs230 may be a powerful tool for malaria control. As a first step, Pfs230 has been expressed in E. coli as a series of recombinant proteins, fused to maltose binding protein. We have used the fusion proteins to assess cellular and humoral immune responses to Pfs230 in malaria-immune adult Gambian blood donors; responses to the fusion proteins have been compared with responses to native Pfs230. The tetrapeptide repeat region of the molecule appears to be immunodominant for both antibody-producing cells and peripheral blood T cells. We postulate that this may represent a mechanism for immune evasion since the N-terminal repeat region of the molecule is cleaved from the mature protein and shed into the plasma. Responses to fusion proteins representing the seven-cysteine motifs were correlated within individual donors, suggesting that cross-reactive epitopes occur within the motifs. Antibody responses to recombinant proteins were poorly correlated with responses to native Pfs230 suggesting that dominant epitopes of the native protein are not adequately represented in the recombinant proteins. Although prokaryotic expression products may be suitable for induction of cellular immune responses to Pfs230, alternative expression systems may be needed for creation of appropriate B cell epitopes.     

2015—2016年山东省由赤道几内亚输入的恶性疟原虫抗药性基因多态性分析

目的　了解山东省由赤道几内亚输入的恶性疟原虫抗性基因多态性情况。方法　采集2015—2016年山东省由赤道几内亚务工返乡的输入性恶性疟患者血样,提取疟原虫基因组DNA,对恶性疟原虫抗性基因Pfcrt、Pfmdr1、Pfdhfr、Pfdhps、K13进行套式PCR扩增、DNA测序和序列对比分析。结果　全部样本5种抗性基因目的片段均成功扩增和测序。Pfcrt野生型、突变型、混合型分别占72.8%、18.6%、8.6%,突变型全部为CVIET（下划线表示突变位点,下同）;Pfmdr1野生型、突变型、混合型分别占20.0%、61.4%、18.6%,突变型主要为YF和NF;Pfdhfr野生型、突变型、混合型分别占1.4%、98.6%、0,突变型主要为AIRNI;Pfdhps野生型、突变型、混合型分别占1.4%、94.3%、4.3%,突变型主要为SGKAA;Pfdhfr和Pfdhps完全抗性基因型IRNGE占8.6%;1.4%的样本K13基因发生A578S突变。结论　山东省由赤道几内亚输入的恶性疟原虫Pfcrt、Pfmdr1、Pfdhfr、Pfdhps、K13基因均发生不同程度突变;Pfcrt突变型比例较低,Pfmdr1、Pfdhfr、Pfdhps突变型比例较高;检测到K13基因发生A578S突变。     

恶性疟原虫11.1基因3个重复片段的克隆及表达

目的　克隆和表达恶性疟原虫 (Pf) 11 1基因产物中的 3个重复片段 3R、6R和 9R。方法　利用设计的引物从培养的恶性疟原虫 3D7株的基因组DNA中扩增出 3个重复片段。PCR产物被克隆入pT7载体中以进双向测序。测序结果用GENETYX MAC软件进行分析。扩增的片段亚克隆入pET32a(+ )或pET32b(+ ) ,并由IPTG诱导在大肠杆菌BL2 1中表达重组蛋白。结果　用PCR方法成功地扩增出 3R、6R和 9R片段 ,大小分别为 5 5 2、630和44 4bp。测序结果显示 ,3D7株的Pf11 1基因比PaloAlto株的Pf11 1基因多 4个 3AA和 1个 6AA重复单元 ,两原虫株的 3R和 6R片段的同源性分别 92 8%和 95 1%。扩增出的 9R片段含有 13个 9AA重复单元。在BL2 1菌株中表达出三个重组蛋白 ,分子量分别为 45、60和 42kDa。结论　用PCR方法分别获得 3R、6R和 9R重复片段并在大肠杆菌中成功表达。 3D7株的Pf11 1基因与PaloAlto株具有高度同源性     

Mortality and morbidity of diabetes in Papua New Guinea

Summary A 95% follow-up of all known diabetic patients in one racial group in Papua New Guinea was performed. Mortality was high with an average life-span of 4–5 years from the time of diagnosis. Microvascular diabetic complications were detected in over half of the surviving diabetic patients whose average duration of known disease was 3.8 years. At present, diabetes mellitus is both a rapidly fatal and morbid disease in Papua New Guinea.     

Diabetes mellitus in urban and rural communities in Papua New Guinea

Summary Oral glucose tolerance tests (75 g) in 185 urban residents of Port Moresby and 105 ethnically similar rural villagers showed that 15.8% of urban residents had diabetes mellitus (two hour plasma glucose > 11.0 mmol/l and a total of 22% were glucose intolerant (plasma glucose > 9.0 mmol/l), compared with 1.0% and 5.5% in rural people. — Urban men and women were significantly fatter than rural people, but within each population glucose tolerance was not significantly related to weight or to age, although the numbers of old people studied were small. Compared to Australians the Papua New Guinea subjects had a higher fasting plasma insulin (16.5 vs 10.7 U/ ml, p = < 0.05); independent of weight fasting plasma insulin was significantly higher in the rural than urban people studied. After the glucose load, plasma insulin and glucose levels were positively correlated in rural people. In contrast, for the urban group the relation best fitted a quadratic function, with decline in plasma insulin at high levels of glucose. — The prevalence of diabetes in urbanised Melanesians in Papua New Guinea appears similar to other South Pacific countries.     

Measurement of ovalocyte frequency in peripheral blood smears in defining ovalocytosis in Papua New Guinea

Red cell oval morphology is still the only accepted basis for the clinical or epidemiological diagnosis of ovalocytosis. Therefore it is important to know the errors when detecting and counting morphological ovalocytes. In all previous studies of ovalocytosis there was no assessment of the variation which may have occurred in classification due to smearing and staining techniques or the criteria for the diagnosis of ovalocyte morphology; nor was inter or intraobserver variation assessed. We report how different peripheral blood smear methods influence the diagnosis of ovalocytosis in populations in the Madang and East Sepik Provinces in Papua New Guinea. We also examined within and between observer variation in the quantitative assessment of ovalocytosis at ×40 and ×100 microscopy powers. A modified method of making a thin malaria blood smear gave the best preservation of red cell morphology and was adopted for the quantitative ovalocytosis studies. A special haematology smear is unnecessary. Ovalocyte frequency estimations were similar when ×40 and ×100 lenses were used, but ×40 was preferable for assessing morphology. Two observers were consistent in their findings and produced very similar results for the high-quality smears from the planned Madang survey, and rather different results for the smears from the unplanned routine Sepik survey. We conclude that measurement error for ovalocytosis assessment can be quite small and unimportant, minimized by careful planning and quality control. Otherwise measurement error is substantial and threatens validity of classification and grading of ovalocytosis.