Low knowledge of newborn danger signs among pregnant women in Papua New Guinea and implications for health seeking behaviour in early infancy - findings from a longitudinal study.

BACKGROUND: Globally, 2.5 million babies die in the first 28 days of life each year with most of these deaths occurring in low- and middle-income countries. Early recognition of newborn danger signs is important in prompting timely care seeking behaviour. Little is known about women's knowledge of newborn danger signs in Papua New Guinea. This study aims to assess this knowledge gap among a cohort of women in East New Britain Province. METHODS: This study assessed knowledge of newborn danger signs (as defined by the World Health Organization) at three time points from a prospective cohort study of women in East New Britain Province, factors associated with knowledge of danger signs after childbirth were assessed using logistic regression. This study includes quantitative and qualitative interview data from 699 pregnant women enrolled at their first antenatal clinic visit, followed up after childbirth (n = 638) and again at one-month post-partum (n = 599). RESULTS: Knowledge of newborn danger signs was very low. Among the 638 women, only 9.4% knew three newborn danger signs after childbirth and only one knew all four essential danger signs defined by Johns Hopkins University 'Birth Preparedness and Complication Readiness' Index. Higher knowledge scores were associated with higher gravidity, income level, partner involvement in antenatal care, and education. CONCLUSION: Low levels of knowledge of newborn danger signs among pregnant women are a potential obstacle to timely care-seeking in rural Papua New Guinea. Antenatal and postnatal education, and policies that support enhanced education and decision-making powers for women and their families, are urgently needed.

Mycoplasma genitalium and Other Reproductive Tract Infections in Pregnant Women, Papua New Guinea, 2015-2017.

Much about the range of pathogens, frequency of coinfection, and clinical effects of reproductive tract infections (RTIs) among pregnant women remains unknown. We report on RTIs (Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum subspecies pallidum, bacterial vaginosis, and vulvovaginal candidiasis) and other reproductive health indicators in 699 pregnant women in Papua New Guinea during 2015-2017. We found M. genitalium, an emerging pathogen in Papua New Guinea, in 12.5% of participants. These infections showed no evidence of macrolide resistance. In total, 74.1% of pregnant women had >1 RTI; most of these infections were treatable. We detected sexually transmitted infections (excluding syphilis) in 37.7% of women. Our findings showed that syndromic management of infections is greatly inadequate. In total, 98.4% of women had never used barrier contraception. These findings will inform efforts to improve reproductive healthcare in Papua New Guinea.

A Trial of a Triple-Drug Treatment for Lymphatic Filariasis.

BACKGROUND: The World Health Organization has targeted lymphatic filariasis for global elimination by 2020 with a strategy of mass drug administration. This trial tested whether a single dose of a three-drug regimen of ivermectin plus diethylcarbamazine plus albendazole results in a greater sustained clearance of microfilariae than a single dose of a two-drug regimen of diethylcarbamazine plus albendazole and is noninferior to the two-drug regimen administered once a year for 3 years. METHODS: In a randomized, controlled trial involving adults from Papua New Guinea with Wuchereria bancrofti microfilaremia, we assigned 182 participants to receive a single dose of the three-drug regimen (60 participants), a single dose of the two-drug regimen (61 participants), or the two-drug regimen once a year for 3 years (61 participants). Clearance of microfilariae from the blood was measured at 12, 24, and 36 months after trial initiation. RESULTS: The three-drug regimen cleared microfilaremia in 55 of 57 participants (96%) at 12 months, in 52 of 54 participants (96%) at 24 months, and in 55 of 57 participants (96%) at 36 months. A single dose of the two-drug regimen cleared microfilaremia in 18 of 56 participants (32%) at 12 months, in 31 of 55 participants (56%) at 24 months, and in 43 of 52 participants (83%) at 36 months (P=0.02 for the three-drug regimen vs. a single dose of the two-drug regimen at 36 months). The two-drug regimen administered once a year for 3 years cleared microfilaremia in 20 of 59 participants (34%) at 12 months, in 42 of 56 participants (75%) at 24 months, and in 51 of 52 participants (98%) at 36 months (P=0.004 for noninferiority of the three-drug regimen vs. the two-drug regimen administered once a year for 3 years at 36 months). Moderate adverse events were more common in the group that received the three-drug regimen than in the combined two-drug-regimen groups (27% vs. 5%, P<0.001). There were no serious adverse events. CONCLUSIONS: The three-drug regimen induced clearance of microfilariae from the blood for 3 years in almost all participants who received the treatment and was superior to the two-drug regimen administered once and noninferior to the two-drug regimen administered once a year for 3 years. (Funded by the Bill and Melinda Gates Foundation; ClinicalTrials.gov number, NCT01975441 .).

Spatial prediction of malaria prevalence in Papua New Guinea: a comparison of Bayesian decision network and multivariate regression modelling approaches for improved accuracy in prevalence prediction.

BACKGROUND: Considerable progress towards controlling malaria has been made in Papua New Guinea through the national malaria control programme's free distribution of long-lasting insecticidal nets, improved diagnosis with rapid diagnostic tests and improved access to artemisinin combination therapy. Predictive prevalence maps can help to inform targeted interventions and monitor changes in malaria epidemiology over time as control efforts continue. This study aims to compare the predictive performance of prevalence maps generated using Bayesian decision network (BDN) models and multilevel logistic regression models (a type of generalized linear model, GLM) in terms of malaria spatial risk prediction accuracy. METHODS: Multilevel logistic regression models and BDN models were developed using 2010/2011 malaria prevalence survey data collected from 77 randomly selected villages to determine associations of Plasmodium falciparum and Plasmodium vivax prevalence with precipitation, temperature, elevation, slope (terrain aspect), enhanced vegetation index and distance to the coast. Predictive performance of multilevel logistic regression and BDN models were compared by cross-validation methods. RESULTS: Prevalence of P. falciparum, based on results obtained from GLMs was significantly associated with precipitation during the 3 driest months of the year, June to August (ß = 0.015; 95% CI = 0.01-0.03), whereas P. vivax infection was associated with elevation (ß = - 0.26; 95% CI = - 0.38 to - 3.04), precipitation during the 3 driest months of the year (ß = 0.01; 95% CI = - 0.01-0.02) and slope (ß = 0.12; 95% CI = 0.05-0.19). Compared with GLM model performance, BDNs showed improved accuracy in prediction of the prevalence of P. falciparum (AUC = 0.49 versus 0.75, respectively) and P. vivax (AUC = 0.56 versus 0.74, respectively) on cross-validation. CONCLUSIONS: BDNs provide a more flexible modelling framework than GLMs and may have a better predictive performance when developing malaria prevalence maps due to the multiple interacting factors that drive malaria prevalence in different geographical areas. When developing malaria prevalence maps, BDNs may be particularly useful in predicting prevalence where spatial variation in climate and environmental drivers of malaria transmission exists, as is the case in Papua New Guinea.

The epidemiology of Plasmodium falciparum and Plasmodium vivax in East Sepik Province, Papua New Guinea, pre- and post-implementation of national malaria control efforts.

BACKGROUND: In the past decade, national malaria control efforts in Papua New Guinea (PNG) have received renewed support, facilitating nationwide distribution of free long-lasting insecticidal nets (LLINs), as well as improvements in access to parasite-confirmed diagnosis and effective artemisinin-combination therapy in 2011-2012. METHODS: To study the effects of these intensified control efforts on the epidemiology and transmission of Plasmodium falciparum and Plasmodium vivax infections and investigate risk factors at the individual and household level, two cross-sectional surveys were conducted in the East Sepik Province of PNG; one in 2005, before the scale-up of national campaigns and one in late 2012-early 2013, after 2 rounds of LLIN distribution (2008 and 2011-2012). Differences between studies were investigated using Chi square (χ2), Fischer's exact tests and Student's t-test. Multivariable logistic regression models were built to investigate factors associated with infection at the individual and household level. RESULTS: The prevalence of P. falciparum and P. vivax in surveyed communities decreased from 55% (2005) to 9% (2013) and 36% to 6%, respectively. The mean multiplicity of infection (MOI) decreased from 1.8 to 1.6 for P. falciparum (p = 0.08) and from 2.2 to 1.4 for P. vivax (p < 0.001). Alongside these reductions, a shift towards a more uniform distribution of infections and illness across age groups was observed but there was greater heterogeneity across the study area and within the study villages. Microscopy positive infections and clinical cases in the household were associated with high rate infection households (> 50% of household members with Plasmodium infection). CONCLUSION: After the scale-up of malaria control interventions in PNG between 2008 and 2012, there was a substantial reduction in P. falciparum and P. vivax infection rates in the studies villages in East Sepik Province. Understanding the extent of local heterogeneity in malaria transmission and the driving factors is critical to identify and implement targeted control strategies to ensure the ongoing success of malaria control in PNG and inform the development of tools required to achieve elimination. In household-based interventions, diagnostics with a sensitivity similar to (expert) microscopy could be used to identify and target high rate households.

Repeated mosquito net distributions, improved treatment, and trends in malaria cases in sentinel health facilities in Papua New Guinea.

BACKGROUND: Long-lasting insecticidal nets (LLIN), improved diagnosis and artemisinin-based combination therapy (ACT) have reduced malaria prevalence in Papua New Guinea since 2008. Yet, national incidence trends are inconclusive due to confounding effects of the scale-up of rapid diagnostic tests, and inconsistencies in routine reporting. METHODS: Malaria trends and their association with LLIN and ACT roll-out between 2010 and 2014 in seven sentinel health facilities were analysed. The analysis included 35,329 fever patients. Intervention effects were estimated using regression models. RESULTS: Malaria incidence initially ranged from 20 to 115/1000 population; subsequent trends varied by site. Overall, LLIN distributions had a cumulative effect, reducing the number of malaria cases with each round (incidence rate ratio ranging from 0.12 to 0.53 in five sites). No significant reduction was associated with ACT introduction. Plasmodium falciparum remained the dominant parasite in all sentinel health facilities. Resurgence occurred in one site in which a shift to early and outdoor biting of anophelines had previously been documented. CONCLUSIONS: LLINs, but not ACT, were associated with reductions of malaria cases in a range of settings, but sustainability of the gains appear to depend on local factors. Malaria programmes covering diverse transmission settings such as Papua New Guinea must consider local heterogeneity when choosing interventions and ensure continuous monitoring of trends.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity.

Iron deficiency during pregnancy is associated with a reduced risk of adverse birth outcomes in a malaria-endemic area in a longitudinal cohort study.

BACKGROUND: Low birth weight (LBW) and preterm birth (PTB) are major contributors to infant mortality and chronic childhood morbidity. Understanding factors that contribute to or protect against these adverse birth outcomes is an important global health priority. Anaemia and iron deficiency are common in malaria-endemic regions, but there are concerns regarding the value of iron supplementation among pregnant women in malaria-endemic areas due to reports that iron supplementation may increase the risk of malaria. There is a lack of evidence on the impact of iron deficiency on pregnancy outcomes in malaria-endemic regions. METHODS: We determined iron deficiency in a cohort of 279 pregnant women in a malaria-endemic area of Papua New Guinea. Associations with birth weight, LBW and PTB were estimated using linear and logistic regression. A causal model using sequential mediation analyses was constructed to assess the association between iron deficiency and LBW, either independently or mediated through malaria and/or anaemia. RESULTS: Iron deficiency in pregnant women was common (71% at enrolment) and associated with higher mean birth weights (230 g; 95% confidence interval, CI 118, 514; p < 0.001), and reduced odds of LBW (adjusted odds ratio, aOR = 0.32; 95% CI 0.16, 0.64; p = 0.001) and PTB (aOR = 0.57; 95% CI 0.30, 1.09; p = 0.089). Magnitudes of effect were greatest in primigravidae (birth weight 351 g; 95% CI 188, 514; p < 0.001; LBW aOR 0.26; 95% CI 0.10, 0.66; p = 0.005; PTB aOR = 0.39, 95% CI 0.16, 0.97; p = 0.042). Sequential mediation analyses indicated that the protective association of iron deficiency on LBW was mainly mediated through mechanisms independent of malaria or anaemia. CONCLUSIONS: Iron deficiency was associated with substantially reduced odds of LBW predominantly through malaria-independent protective mechanisms, which has substantial implications for understanding risks for poor pregnancy outcomes and evaluating the benefit of iron supplementation in pregnancy. This study is the first longitudinal study to demonstrate a temporal relationship between antenatal iron deficiency and improved birth outcomes. These findings suggest that iron supplementation needs to be integrated with other strategies to prevent or treat infections and undernutrition in pregnancy to achieve substantial improvements in birth outcomes.

The impact of the scale-up of malaria rapid diagnostic tests on the routine clinical diagnosis procedures for febrile illness: a series of repeated cross-sectional studies in Papua New Guinea.

BACKGROUND: This paper examines the impact of the scale-up of malaria rapid diagnostic tests (RDT) on routine clinical diagnosis procedures for febrile illness in primary healthcare settings in Papua New Guinea. METHODS: Repeat, cross-sectional surveys in randomly selected primary healthcare services were conducted. Surveys included passive observation of consecutive febrile case management cases and were completed immediately prior to RDT scale-up (2011) and at 12- (2012) and 60-months (2016) post scale-up. The frequency with which specified diagnostic questions and procedures were observed to occur, with corresponding 95% CIs, was calculated for febrile patients prescribed anti-malarials pre- and post-RDT scale-up and between febrile patients who tested either negative or positive for malaria infection by RDT (post scale-up only). RESULTS: A total of 1809 observations from 120 health facilities were completed across the three survey periods of which 915 (51%) were prescribed an anti-malarial. The mean number of diagnostic questions and procedures asked or performed, leading to anti-malarial prescription, remained consistent pre- and post-RDT scale-up (range 7.4-7.7). However, alterations in diagnostic content were evident with the RDT replacing body temperature as the primary diagnostic procedure performed (observed in 5.3 and 84.4% of cases, respectively, in 2011 vs. 77.9 and 58.2% of cases in 2016). Verbal questioning, especially experience of fever, cough and duration of symptoms, remained the most common feature of a diagnostic examination leading to anti-malarial prescription irrespective of RDT use (observed in 96.1, 86.8 and 84.8% of cases, respectively, in 2011 vs. 97.5, 76.6 and 85.7% of cases in 2016). Diagnostic content did not vary substantially by RDT result. CONCLUSIONS: Rapid diagnostic tests scale-up has led to a reduction in body temperature measurement. Investigations are very limited when malaria infection is ruled out as a cause of febrile illness by RDT.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated malaria in Papua New Guinea.

BACKGROUND: In 2009, the Papua New Guinea (PNG) Department of Health adopted artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PPQ) as the first- and second-line treatments for uncomplicated malaria, respectively. This study was conducted to assess the efficacy of both drugs following adoption of the new policy. METHODS: Between June 2012 and September 2014, a therapeutic efficacy study was conducted in East Sepik and Milne Bay Provinces of PNG in accordance with the standard World Health Organization (WHO) protocol for surveillance of anti-malarial drug efficacy. Patients ≥ 6 months of age with microscopy confirmed Plasmodium falciparum or Plasmodium vivax mono-infections were enrolled, treated with AL or DHA-PPQ, and followed up for 42 days. Study endpoints were adequate clinical and parasitological response (ACPR) on days 28 and 42. The in vitro efficacy of anti-malarials and the prevalence of selected molecular markers of resistance were also determined. RESULTS: A total of 274 P. falciparum and 70 P. vivax cases were enrolled. The day-42 PCR-corrected ACPR for P. falciparum was 98.1% (104/106) for AL and 100% (135/135) for DHA-PPQ. The day-42 PCR-corrected ACPR for P. vivax was 79.0% (15/19) for AL and 92.3% (36/39) for DHA-PPQ. Day 3 parasite clearance of P. falciparum was 99.2% with AL and 100% with DHA-PPQ. In vitro testing of 96 samples revealed low susceptibility to chloroquine (34% of samples above IC50 threshold) but not to lumefantrine (0%). Molecular markers assessed in a sub-set of the study population indicated high rates of chloroquine resistance in P. falciparum (pfcrt SVMNT: 94.2%, n = 104) and in P. vivax (pvmdr1 Y976F: 64.8%, n = 54). CONCLUSIONS: AL and DHA-PPQ were efficacious as first- and second-line treatments for uncomplicated malaria in PNG. Continued in vivo efficacy monitoring is warranted considering the threat of resistance to artemisinin and partner drugs in the region and scale-up of artemisinin-based combination therapy in PNG.

Mosquito Behavior Change After Distribution of Bednets Results in Decreased Protection Against Malaria Exposure.

Background: Behavioral resilience in mosquitoes poses a significant challenge to mosquito control. Although behavior changes in anopheline vectors have been reported over the last decade, there are no empirical data to suggest they compromise the efficacy of vector control in reducing malaria transmission. Methods: In this study, we quantified human exposure to both bites and infective bites of a major malaria vector in Papua New Guinea over the course of 4 years surrounding nationwide bednet distribution. We also quantified malaria infection prevalence in the human population during the same time period. Results: We observed a shift in mosquito biting to earlier hours of the evening, before individuals are indoors and protected by bednets, followed by a return to preintervention biting rates. As a result, net users and non-net users experienced higher levels of transmission than before the intervention. The personal protection provided by a bednet decreased over the study period and was lowest in the adult population, who may be an important reservoir for transmission. Malaria prevalence decreased in only 1 of 3 study villages after the distribution. Discussion: This study highlights the necessity of validating and deploying vector control measures targeting outdoor exposure to control and eliminate malaria.

Optimal Antimalarial Dose Regimens for Sulfadoxine-Pyrimethamine with or without Azithromycin in Pregnancy Based on Population Pharmacokinetic Modeling.

Optimal dosing of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment in pregnancy remains to be established, particularly when coadministered with azithromycin (AZI). To further characterize SP pharmacokinetics in pregnancy, plasma concentration-time data from 45 nonpregnant and 45 pregnant women treated with SP-AZI (n = 15 in each group) and SP-chloroquine (n = 30 in each group) were analyzed. Population nonlinear mixed-effect pharmacokinetic models were developed for pyrimethamine (PYR), sulfadoxine (SDOX), and N-acetylsulfadoxine (the SDOX metabolite NASDOX), and potential covariates were included. Pregnancy increased the relative clearance (CL/F) of PYR, SDOX, and NASDOX by 48, 29, and 70%, respectively, as well as the relative volumes of distribution (V/F) of PYR (46 and 99%) and NASDOX (46%). Coadministration of AZI resulted in a greater increase in PYR CL/F (80%) and also increased NASDOX V/F by 76%. Apparent differences between these results and those of published studies of SP disposition may reflect key differences in study design, including the use of an early postpartum follow-up study rather than a nonpregnant comparator group. Simulations based on the final population model demonstrated that, compared to conventional single-dose SP in nonpregnant women, two such doses given 24 h apart should ensure that pregnant women have similar drug exposure, while three daily SP doses may be required if SP is given with AZI. The results of past and ongoing trials using recommended adult SP doses with or without AZI in pregnant women may need to be interpreted in light of these findings and consideration given to using increased doses in future trials.

Synergistic effect of IL-12 and IL-18 induces TIM3 regulation of γδ T cell function and decreases the risk of clinical malaria in children living in Papua New Guinea.

BACKGROUND: γδ T cells are important for both protective immunity and immunopathogenesis during malaria infection. However, the immunological processes determining beneficial or detrimental effects on disease outcome remain elusive. The aim of this study was to examine expression and regulatory effect of the inhibitory receptor T-cell immunoglobulin domain and mucin domain 3 (TIM3) on γδ T cells. While TIM3 expression and function on conventional αß T cells have been clearly defined, the equivalent characterization on γδ T cells and associations with disease outcomes is limited. This study investigated the functional capacity of TIM3+ γδ T cells and the underlying mechanisms contributing to TIM3 upregulation and established an association with malaria disease outcomes. METHODS: We analyzed TIM3 expression on γδ T cells in 132 children aged 5-10 years living in malaria endemic areas of Papua New Guinea. TIM3 upregulation and effector functions of TIM3+ γδ T cells were assessed following in vitro stimulation with parasite-infected erythrocytes, phosphoantigen and/or cytokines. Associations between the proportion of TIM3-expressing cells and the molecular force of infection were tested using negative binomial regression and in a Cox proportional hazards model for time to first clinical episode. Multivariable analyses to determine the association of TIM3 and IL-18 levels were conducted using general linear models. Malaria infection mouse models were utilized to experimentally investigate the relationship between repeated exposure and TIM3 upregulation. RESULTS: This study demonstrates that even in the absence of an active malaria infection, children of malaria endemic areas have an atypical population of TIM3-expressing γδ T cells (mean frequency TIM3+ of total γδ T cells 15.2% ± 12). Crucial factors required for γδ T cell TIM3 upregulation include IL-12/IL-18, and plasma IL-18 was associated with TIM3 expression (P = 0.002). Additionally, we show a relationship between TIM3 expression and infection with distinct parasite clones during repeated exposure. TIM3+ γδ T cells were functionally impaired and were associated with asymptomatic malaria infection (hazard ratio 0.54, P = 0.032). CONCLUSIONS: Collectively our data demonstrate a novel role for IL-12/IL-18 in shaping the innate immune response and provide fundamental insight into aspects of γδ T cell immunoregulation. Furthermore, we show that TIM3 represents an important γδ T cell regulatory component involved in minimizing malaria symptoms.

Insecticide-treated nets and malaria prevalence, Papua New Guinea, 2008-2014.

OBJECTIVE: To investigate changes in malaria prevalence in Papua New Guinea after the distribution of long-lasting Insecticide-treated nets, starting in 2004, and the introduction of artemisinin-based combination therapy in 2011. METHODS: Two malaria surveys were conducted in 2010-2011 and 2013-2014. They included 77 and 92 randomly selected villages, respectively. In each village, all members of 30 randomly selected households gave blood samples and were assessed for malaria infection by light microscopy. In addition, data were obtained from a malaria survey performed in 2008-2009. RESULTS: The prevalence of malaria below 1600 m in altitude decreased from 11.1% (95% confidence interval, CI: 8.5-14.3) in 2008-2009 to 5.1% (95% CI 3.6-7.4) in 2010-2011 and 0.9% (95% CI 0.6-1.5) in 2013-2014. Prevalence decreased with altitude. Plasmodium falciparum was more common than P. vivax overall, but not everywhere, and initially the prevalence of P. vivax infection decreased more slowly than P. falciparum infection. Malaria infections were clustered in households. In contrast to findings in 2008-2009, no significant association between net use and prevalence was found in the later two surveys. The prevalence of both fever and splenomegaly also decreased but their association with malaria infection became stronger. CONCLUSION: Large-scale insecticide-treated net distribution was associated with an unprecedented decline in malaria prevalence throughout Papua New Guinea, including epidemic-prone highland areas. The decline was accompanied by broader health benefits, such as decreased morbidity. Better clinical management of nonmalarial fever and research into residual malaria transmission are required.

Proinflammatory responses and higher IL-10 production by T cells correlate with protection against malaria during pregnancy and delivery outcomes.

Pregnancy triggers immunological changes aimed to tolerate the fetus. However, it has not been properly addressed whether similar changes occur in tropical areas with high infection pressure and whether these changes render women more susceptible to infectious diseases. We compared the frequencies of T cell subsets, including regulatory T cells, in pregnant and nonpregnant women from Papua New Guinea, a high malaria transmission area, and from Spain, a malaria-free country. We also assessed the relationship among these cellular subsets, malaria infection, and delivery outcomes. CD4(+)FOXP3(+)CD127(low) T cells (Tregs) were decreased in pregnant women in both countries but were not associated with malaria infection or poor delivery outcomes. An expansion of IFN-γ-producing cells and intracytoplasmic IFN-γ levels was found in pregnant compared with nonpregnant women only in Papua New Guinea. Increased CD4(+)IL-10(+)IFN-γ(+) frequencies and Treg-IFN-γ production were found in women with current Plasmodium falciparum infection. Higher CD4(+)IL-10(-)IFN-γ(+) T cells frequencies and production of proinflammatory cytokines (including TNF and IL-2) at recruitment (first antenatal visit) had a protective association with birth weight and future (delivery) P. falciparum infection, respectively. Higher intracellular IL-10 levels in T cells had a protective association with future P. falciparum infection and hemoglobin levels at delivery. The protective associations were found also with nonmalaria-specific T cell responses. Treg frequencies positively correlated with plasma eotaxin concentrations, but this subset did not express eotaxin receptor CCR3. Thus, an activated immune system during pregnancy might contribute to protection against malaria during pregnancy and poor delivery outcomes.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

The treatment of non-malarial febrile illness in Papua New Guinea: findings from cross sectional and longitudinal studies of health worker practice.

BACKGROUND: The Papua New Guinea Department of Health recently shifted from a presumptive to a 'test and treat' malaria case management policy. This shift was supported by the widespread introduction of malaria rapid diagnostic tests in health facilities across the country. Health workers received training and job-aids detailing how to conduct and interpret a malaria rapid diagnostic test and how to treat test positive cases; however, little instruction on treating non-malaria febrile cases was provided. Accordingly, this study examined health worker case management of non-malarial febrile patients in the 12-month period immediately following the introduction of the revised malaria case management policy. METHODS: Data were collected from a country-wide cross-sectional survey of febrile case management at randomly selected health facilities and from longitudinal surveillance at sentinel health facilities. Analysis was restricted to febrile patients who tested negative for malaria infection by rapid diagnostic test (N=303 and 5705 outpatients, respectively). RESULTS AND DISCUSSION: 96.8% of non-malarial febrile patients received a diagnosis in the longitudinal sample, compared to 52.4% of the cross-sectional sample. Respiratory tract infections were the most commonly reported diagnoses. Over 90% of patients in both samples were prescribed one or more medications, most commonly an analgesic (71.3 & 72.9% of the longitudinal and cross-sectional samples, respectively), some form of antibiotic (72.7 & 73.4%, respectively) and/or an anthelminthic (17.9 & 16.5%, respectively). Prescribing behaviour was adherent with the recommendations in the standard treatment guidelines in fewer than 20% of cases (longitudinal sample only). CONCLUSION: Many non-malarial febrile patients are not provided with a diagnosis. When diagnoses are provided they are typically some form of respiratory tract infection. Antibiotics and analgesics are widely prescribed, although medications prescribed rarely adhere to the Papua New Guinea standard treatment guidelines. These findings indicate that Papua New Guinea health workers require support for non-malarial febrile illness case management.

Different Regions of Plasmodium falciparum Erythrocyte-Binding Antigen 175 Induce Antibody Responses to Infection of Varied Efficacy.

BACKGROUND: Increasing evidence suggests that antibodies against merozoite proteins involved in Plasmodium falciparum invasion into the red blood cell play an important role in clinical immunity to malaria. Erythrocyte-binding antigen 175 (EBA-175) is the best-characterized P. falciparum invasion ligand, reported to recognize glycophorin A on the surface of erythrocytes. Its protein structure comprises 6 extracellular regions. Whereas region II contains Duffy binding-like domains involved in the binding to glycophorin A, the functional role of regions III-V is less clear. METHODS: We developed a novel cytometric bead array for assessment of antigen-specific antibody concentration in plasma to evaluate the efficacy of immune responses to different regions of EBA-175 and associations between antibody levels with protection from symptomatic malaria in a treatment-reinfection cohort study. RESULTS: We found that while antibodies to region II are highly abundant, circulating levels as low as 5-10 µg/mL of antibodies specific for region III or the highly conserved regions IV-V predict strong protection from clinical malaria. CONCLUSIONS: These results lend support for the development of conserved regions of EBA-175 as components in a combination of a malaria vaccine.

Impact of Placental Malaria and Hypergammaglobulinemia on Transplacental Transfer of Respiratory Syncytial Virus Antibody in Papua New Guinea.

BACKGROUND: Passively acquired respiratory syncytial virus (RSV) neutralizing antibody protects against RSV-associated lower respiratory infections, but placental malaria (PM) and maternal hypergammaglobulinemia might interfere with transplacental immunoglobulin transport. METHODS: We measured RSV plaque-reduction neutralization (PRN) antibody in 300 full-term maternal/cord serum pairs in 2 cohorts in malaria-endemic Papua New Guinea: Alexishafen (2005-2008) and the Fetal Immunity Study (FIS) (2011-2013). We defined impaired transport as a cord-to-maternal titer ratio <1.0 and a protective RSV PRN titer (PRNT) ≥1:200. RESULTS: PM and hypergammaglobulinemia occurred in 60% and 54% of Alexishafen mothers versus 8% and 9% of FIS mothers, respectively. 34% of Alexishafen and 32% of FIS pairs demonstrated impaired transport. Multivariate modeling revealed significant associations between increasing maternal IgG (log2) and impaired transport (adjusted OR, Alexishafen: 2.68 [1.17-6.14], FIS: 6.94 [1.94-24.8]) but no association with PM. 34% of Alexishafen and 31% of FIS cord PRNTs were <1:200. CONCLUSIONS: Impaired RSV antibody transport was observed in approximately one-third of maternal/cord pairs. Hypergammaglobulinemia, but not PM, was associated with impaired transport, particularly among women with low RSV PRNT. Detection of RSV PRNT <1:200 in one-third of cord sera confirms the need to increase levels of RSV neutralizing antibody in pregnant women through maternal immunization.

Efficacy, Safety, and Pharmacokinetics of Coadministered Diethylcarbamazine, Albendazole, and Ivermectin for Treatment of Bancroftian Filariasis.

BACKGROUND: Available treatments for lymphatic filariasis (LF) are limited in their longterm clearance of microfilaria from the blood. The safety and efficacy of a single-dose triple-drug therapy of the antifilarial drugs diethylcarbamazine (DEC), ivermectin (IVM), and albendazole (ALB) for LF are unknown. METHODS: We performed a pilot study to test the efficacy, safety, and pharmacokinetics of single-dose DEC, IVM, and ALB in Wuchereria bancrofti-infected Papua New Guineans. Adults were randomized into 2 treatment arms, DEC 6 mg/kg + ALB 400 mg (N = 12) or DEC 6 mg/kg + ALB 400 mg + IVM 200 µg/kg (N = 12), and monitored for microfilaria, parasite antigenemia, adverse events (AEs), and serum drug levels. RESULTS: Triple-drug therapy induced >2-log reductions in microfilaria levels at 36 and 168 hours after treatment compared with approximately 1-log reduction with 2 drugs. All 12 individuals who received 3 drugs were microfilaria negative 1 year after treatment, whereas 11 of 12 individuals in the 2-drug regimen were microfilaria positive. In 6 participants followed 2 years after treatment, those who received 3 drugs remained microfilaria negative. AEs, particularly fever, myalgias, pruritus, and proteinuria/hematuria, occurred in 83% vs 50% of those receiving triple-drug compared to 2-drug treatment respectively (P = .021); all resolved within 7 days after treatment. No serious AEs were observed in either group. There was no significant effect of IVM on DEC or ALB drug levels. CONCLUSIONS: Triple-drug therapy is safe and more effective than DEC + ALB for Bancroftian filariasis and has the potential to accelerate elimination of lymphatic filariasis. CLINICAL TRIALS REGISTRATION: NCT01975441.