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Abstract Background Members of the Anopheles punctulatus group (AP group) are the primary vectors of human malaria in Papua New Guinea. The AP group includes 13 sibling species, most of them morphologically indistinguishable. Understanding why only certain species are able to transmit malaria requires a better comprehension of their evolutionary history. In particular, understanding relationships and divergence times among Anopheles species may enable assessing how malaria-related traits (e.g. blood feeding behaviours, vector competence) have evolved. Methods DNA sequences of 14 mitochondrial (mt) genomes from five AP sibling species and two species of the Anopheles dirus complex of Southeast Asia were sequenced. DNA sequences from all concatenated protein coding genes (10,770 bp) were then analysed using a Bayesian approach to reconstruct phylogenetic relationships and date the divergence of the AP sibling species. Results Phylogenetic reconstruction using the concatenated DNA sequence of all mitochondrial protein coding genes indicates that the ancestors of the AP group arrived in Papua New Guinea 25 to 54 million years ago and rapidly diverged to form the current sibling species. Conclusion Through evaluation of newly described mt genome sequences, this study has revealed a divergence among members of the AP group in Papua New Guinea that would significantly predate the arrival of humans in this region, 50 thousand years ago. The divergence observed among the mtDNA sequences studied here may have resulted from reproductive isolation during historical changes in sea-level through glacial minima and maxima. This leads to a hypothesis that the AP sibling species have evolved independently for potentially thousands of generations. This suggests that the evolution of many phenotypes, such as insecticide resistance will arise independently in each of the AP sibling species studied here.

Abstract Background Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. Results Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; Isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; Streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. Conclusions We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.

Abstract Background Male circumcision (MC) has been shown to reduce the risk of HIV acquisition among heterosexual men, with WHO recommending MC as an essential component of comprehensive HIV prevention programs in high prevalence settings since 2007. While Papua New Guinea (PNG) has a current prevalence of only 1%, the high rates of sexually transmissible diseases and the extensive, but unregulated, practice of penile cutting in PNG have led the National Department of Health (NDoH) to consider introducing a MC program. Given public interest in circumcision even without active promotion by the NDoH, examining the potential health systems implications for MC without raising unrealistic expectations presents a number of methodological issues. In this study we examined health systems lessons learned from a national no-scalpel vasectomy (NSV) program, and their implications for a future MC program in PNG. Methods Fourteen in-depth interviews were conducted with frontline health workers and key government officials involved in NSV programs in PNG over a 3-week period in February and March 2011. Documentary, organizational and policy analysis of HIV and vasectomy services was conducted and triangulated with the interviews. All interviews were digitally recorded and later transcribed. Application of the WHO six building blocks of a health system was applied and further thematic analysis was conducted on the data with assistance from the analysis software MAXQDA. Results Obstacles in funding pathways, inconsistent support by government departments, difficulties with staff retention and erratic delivery of training programs have resulted in mixed success of the national NSV program. Conclusions In an already vulnerable health system significant investment in training, resources and negotiation of clinical space will be required for an effective MC program. Focused leadership and open communication between provincial and national government, NGOs and community is necessary to assist in service sustainability. Ensuring clear policy and guidance across the entire sexual and reproductive health sector will provide opportunities to strengthen key areas of the health system.

Abstract Background Male circumcision (MC) has been shown to reduce vaginal transmission of HIV to men. While community acceptability is important in a countries preparedness to introduce MC, it is equally important to map contemporary MC and other penile cutting practices, and the socio-cultural dimensions underpinning these practices. Methods A total of 482 men and women (n = 276 and n = 210, respectively) participated in 82 semi-structured and 45 focus group discussions from four different provinces of Papua New Guinea (PNG), each representing one of the four socially and geographically diverse regions of the country. Results Of the men interviewed 131 self-reported that they had undergone a penile alteration with some reporting multiple types. Practices were diverse and could be grouped into five broad categories: traditional (customary) penile cutting; contemporary penile cutting; medical circumcision; penile inserts; and penile bloodletting practices in which sharp objects are used to incise the glans and or inserted and withdrawn from the male urethra or in order to induce bleeding. Socio-cultural traditions, enhanced sexual pleasure and improved genital hygiene were key motivators for all forms of penile practices. Conclusions The findings from this study highlight the complex and diverse nature of penile practices in PNG and their association with notions of masculinity, sexuality and contagion. Contemporary penile practices are critical to a community’s acceptance of MC and of a country’s ability to successfully implement MC in the context of a rich and dynamic culture of penile practices. If a MC program were to be successfully rolled out in PNG to prevent HIV it would need to work within and build upon these diverse cultural meanings and motivators for penile practices already commonly performed in PNG by men.

Abstract Background Papua New Guinea (PNG) is a highly malaria endemic country in the South-West Pacific with a population of approximately 6.6 million (2009). In 2004, the country intensified its malaria control activities with support from the Global Fund. With the aim of achieving 80% ownership and usage, a country-wide campaign distributed two million free long-lasting insecticide-treated nets (LLINs). Methods In order to evaluate outcomes of the campaign against programme targets, a country-wide household survey based on stratified multi-stage random sampling was carried out in 17 of the 20 provinces after the campaign in 2008/09. In addition, a before-after assessment was carried out in six purposively selected sentinel sites. A structured questionnaire was administered to the heads of sampled households to elicit net ownership and usage information. Results After the campaign, 64.6% of households owned a LLIN, 80.1% any type of mosquito net. Overall usage by household members amounted to 32.5% for LLINs and 44.3% for nets in general. Amongst children under five years, 39.5% used a LLIN and 51.8% any type of net, whereas 41.3% of pregnant women used a LLIN and 56.1% any net. Accessibility of villages was the key determinant of net ownership, while usage was mainly determined by ownership. Most (99.5%) of the household members who did not sleep under a net did not have access to a (unused) net in their household. In the sentinel sites, LLIN ownership increased from 9.4% to 88.7%, ownership of any net from 52.7% to 94.1%. Usage of LLINs increased from 5.5% to 55.1%, usage of any net from 37.3% to 66.7%. Among children under five years, usage of LLINs and of nets in general increased from 8.2% to 67.0% and from 44.6% to 76.1%, respectively (all p ≤ 0.001). Conclusions While a single round of free distribution of LLINs significantly increased net ownership, an insufficient number of nets coupled with a heterogeneous distribution led to overall low usage rates. Programme targets were missed mainly as a result of the distribution mechanism itself and operational constraints in this very challenging setting.

Abstract Background This study aimed to document malaria case management practices in Papua New Guinea prior to the introduction of a revised national malaria treatment protocol. The revised protocol stipulates routine testing of malaria infection by rapid diagnostic test or microscopy, anti-malarial prescription to test positive cases only, and the introduction of a new artemisinin-based first-line anti-malarial. Findings presented in this paper primarily focus on diagnostic, prescription and treatment counselling practices. Methods In a national cross-sectional survey of 79 randomly selected health facilities, data were collected via non-participant observation of the clinical case management of patients presenting with fever or a recent history of fever. Data were recorded on a structured clinical observation instrument. Results Overall, 15% of observed fever patients (n = 468) were tested for malaria infection by rapid diagnostic test and a further 3.6% were tested via microscopy. An anti-malarial prescription was made in 96.4% (451/468) of cases, including 100% (17/17) of test positive cases and 82% (41/50) of test negative cases. In all, 79.8% of anti-malarial prescriptions conformed to the treatment protocol current at the time of data collection. The purpose of the prescribed medication was explained to patients in 63.4% of cases, dosage/regimen instructions were provided in 75.7% of cases and the possibility of adverse effects and what they might look like were discussed in only 1.1% of cases. Conclusion The revised national malaria treatment protocol will require a substantial change in current clinical practice if it is to be correctly implemented and adhered to. Areas that will require the most change include the shift from presumptive to RDT/microscopy confirmed diagnosis, prescribing (or rather non-prescribing) of anti-malarials to patients who test negative for malaria infection, and the provision of thorough treatment counselling. A comprehensive clinician support programme, possibly inclusive of ‘booster’ training opportunities and regular clinical supervision will be needed to support the change.

Abstract Microvascular sequestration of Plasmodium falciparum underlies cerebral malaria. Despite suggestive ex vivo evidence, this phenomenon has not been convincingly demonstrated in coma complicating Plasmodium vivax malaria. Severely-ill Papua New Guinean children with mixed P. falciparum/P. vivax infections are more likely to develop cerebral malaria and die than those with P. falciparum alone, possibly reflecting P. vivax sequestration. Nested PCR was performed on post mortem brain tissue from three such children dying from cerebral malaria due to mixed-species infections. No P. vivax DNA was detected. These findings do not support the hypothesis that P. vivax sequestration occurs in human brain.

Abstract Background Adult male surgical circumcision (MC) has been shown to reduce HIV acquisition in men and is recommended by the WHO for inclusion in comprehensive national HIV prevention programs in high prevalence settings. Only limited research to date has been conducted in countries experiencing moderate burden epidemics, where the acceptability, operational feasibility and potential epidemiological impact of MC remain unclear. Methods A multi-method qualitative research study was conducted at four sites in Papua New Guinea (PNG), with 24 focus group discussions and 65 in-depth interviews carried out among 276 men. Results The majority of men were in favour of MC being introduced for HIV prevention in PNG and considered improved genital hygiene, enhanced sexual pleasure and culturally appropriateness key factors in the acceptability of a future intervention. A minority of men were against the introduction of MC, primarily due to concerns regarding sexual risk compensation and that the intervention went against prevailing cultural and religious beliefs. Conclusion This is one of the first community-based MC acceptability studies conducted in a moderate prevalence setting outside of Africa. Research findings from this study suggest that a future MC program for HIV prevention would be widely accepted by men in PNG.

Abstract Background Reports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates. Methods Pvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples. Results Genotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele. Conclusions The LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.

Abstract Background The Plasmodium falciparum merozoite surface protein Pf38 is targeted by antibodies of malaria immune adults and has been shown to be under balancing (immune) selection in a Gambian parasite population, indicating potential as a malaria vaccine candidate. This study explores the population genetics of Pf38 in Papua New Guinea, to determine the extent and geographic distribution of diversity and to measure selective pressure along the length of the gene. Methods Using samples collected during community-based cross-sectional surveys in the Mugil and Wosera regions, the Pf38 genes of 59 P. falciparum isolates were amplified and sequenced. These sequences, along with previously sequenced Gambian and laboratory isolates, were then subjected to an array of population genetic analyses, examining polymorphisms, haplotype diversity and balancing selection. In addition to whole-gene analysis, the two 6-cys domains were considered separately, to investigate domain specific polymorphism and selection. Results Nineteen polymorphic sites were identified in the Pf 38 gene. Of these, 13 were found in the Gambia, 10 in Mugil and 8 in Wosera. Notably, the majority of common polymorphisms were confined to domain I. Although only moderate levels of nucleotide diversity were observed, the haplotype diversity was high in all populations, suggesting extensive recombination. Analyses of the full-length sequence provided only modest evidence for balancing selection. However, there was a strong contrast between domain I, which showed strong evidence for positive balancing selection, and domain II which was neutral. Analyses of the geographic distribution of Pf38 haplotypes showed that four haplotypes accounted for the majority of sequences found world-wide, but there were many more haplotypes unique to the African than the PNG populations. Conclusion This study confirmed previous findings that Pf38 is a polymorphic gene under balancing selection. However, analysing polymorphism and selection across the length of the gene painted a considerably different picture. Domain I is highly polymorphic and the target of significant balancing selection. In contrast, domain II is relatively conserved and does not show evidence of immune selective pressure. The findings have implications for future population genetic studies on vaccine candidates, showing that the biological context must also be considered as a framework for analysis.

Abstract Background A review of the barriers to mosquito net use in malaria-endemic countries has yet to be presented in the published literature despite considerable research interest in this area. This paper partly addresses this gap by reviewing one component of the evidence base; namely, published research pertaining to self-reported reasons for not using a mosquito net among net 'owning' individuals. It was anticipated that the review findings would potentially inform an intervention or range of interventions best suited to promoting greater net use amongst this group. Method Studies were sought via a search of the Medline database. The key inclusion criteria were: that study participants could be identified as owning a mosquito net or having a mosquito net available for use; that these participants on one or more occasions were identified or self-reported as not using the mosquito net; and that reasons for not using the mosquito net were reported. Studies meeting these criteria were included irrespective of mosquito net type. Results A total of 22 studies met the inclusion criteria. Discomfort, primarily due to heat, and perceived (low) mosquito density were the most widely identified reason for non-use. Social factors, such as sleeping elsewhere, or not sleeping at all, were also reported across studies as were technical factors related to mosquito net use (i.e. not being able to hang a mosquito net or finding it inconvenient to hang) and the temporary unavailability of a normally available mosquito net (primarily due to someone else using it). However, confidence in the reported findings was substantially undermined by a range of methodological limitations and a dearth of dedicated research investigation. Conclusions The findings of this review should be considered highly tentative until such time as greater quantities of dedicated, well-designed and reported studies are available in the published literature. The current evidence-base is not sufficient in scope or quality to reliably inform mosquito net promoting interventions or campaigns targeted at individuals who own, but do not (reliably) use, mosquito nets.

Abstract Background Accurate diagnosis of Plasmodium infections is essential for malaria morbidity and mortality reduction in tropical areas. Despite great advantages of light microscopy (LM) for malaria diagnosis, its limited sensitivity is a critical shortfall for epidemiological studies. Robust molecular diagnostics tools are thus needed. Methods The present study describes the development of a duplex quantitative real time PCR (qPCR) assay, which specifically detects and quantifies the four human Plasmodium species. Performance of this method was compared to PCR-ligase detection reaction-fluorescent microsphere assay (PCR_LDR_FMA), nested PCR (nPCR) and LM, using field samples collected from 452 children one to five years of age from the Sepik area in Papua New Guinea. Agreement between diagnostic methods was calcualted using kappa statistics. Results The agreement of qPCR with other molecular diagnostic methods was substantial for the detection of P. falciparum, but was moderate for the detection of P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax prevalence by qPCR was 40.9% and 65.7% respectively. This compares to 43.8% and 73.2% by nPCR and 47.1% and 67.5% by PCR_LDR_FMA. P. malariae and P. ovale prevalence was 4.7% and 7.3% by qPCR, 3.3% and 3.8% by nPCR, and 7.7% and 4.4% by PCR_LDR_FMA. Prevalence by LM was lower for all four species, being 25.4% for P. falciparum, 54.9% for P. vivax, 2.4% for P. malariae and 0.0% for P. ovale. The quantification by qPCR closely correlated with microscopic quantification for P. falciparum and P. vivax samples (R2 = 0.825 and R2 = 0.505, respectively). The low prevalence of P. malariae and P. ovale did not permit a solid comparative analysis of quantification for these species. Conclusions The qPCR assay developed proved optimal for detection of all four Plasmodium species. Densities by LM were well reflected in quantification results by qPCR, whereby congruence was better for P. falciparum than for P. vivax. This likely is a consequence of the generally lower P. vivax densities. Easy performance of the qPCR assay, a less laborious workflow and reduced risk of contamination, together with reduced costs per sample through reduced reaction volume, opens the possibility to implement qPCR in endemic settings as a suitable diagnostic tool for large epidemiological studies.

Abstract Background Building on previous acceptability research undertaken in sub-Saharan Africa this article aims to investigate the acceptability of intermittent preventive treatment of malaria in infants (IPTi) in Papua New Guinea (PNG). Methods A questionnaire was administered to mothers whose infants participated in the randomised placebo controlled trial of IPTi. Mothers whose infants participated and who refused to participate in the trial, health workers, community reporters and opinion leaders were interviewed. Men and women from the local community also participated in focus group discussions. Results Respondents viewed IPTi as acceptable in light of wider concern for infant health and the advantages of trial participation. Mothers reported complying with at-home administration of IPTi due to perceived benefits of IPTi and pressure from health workers. In spite of patchy knowledge, respondents also demonstrated a demand for infant vaccinations and considered non-vaccination to be neglect. There is little evidence that IPTi has negative impacts on attitudes to EPI, EPI adherence or existing malaria prevention practices. Conclusion The degree of similarity between findings from the acceptability studies undertaken in sub-Saharan Africa and PNG allows some generalization relating to the implementation of IPTi outside of Africa: IPTi fits well with local health cultures, appears to be accepted easily and has little impact on attitudes towards EPI or malaria prevention. The study adds to the evidence indicating that IPTi could be rolled out in a range of social and cultural contexts.

Abstract Background The South West Pacific nation of Papua New Guinea has intense year round transmission of Plasmodium falciparum on the coast and in the low-lying inland areas. Local heterogeneity in the epidemiology of malaria suggests that parasites from multiple locations will need to be surveyed to define the population biology of P. falciparum in the region. This study describes the population genetics of P. falciparum in thirteen villages spread over four distinct catchment areas of Papua New Guinea. Methods Ten microsatellite loci were genotyped in 318 P. falciparum isolates from the parasite populations of two inland catchment areas, namely Wosera (number of villages (n) = 7) and Utu (n = 1) and; and two coastal catchments, Malala (n = 3) and Mugil (n = 3). Analysis of the resultant multilocus haplotypes was done at different spatial scales (2-336 km) to define the genetic diversity (allelic richness and expected heterozygosity), linkage disequilibrium and population structure throughout the study area. Results Although genetic diversity was high in all parasite populations, it was also variable with a lower allelic richness and expected heterozygosity for inland populations compared to those from the more accessible coast. This variability was not correlated with two proxy measures of transmission intensity, the infection prevalence and the proportion multiple infections. Random associations among the microsatellite loci were observed in all four catchments showing that a substantial degree of out-crossing occurs in the region. Moderate to very high levels of population structure were found but the amount of genetic differentiation (FST) did not correlate with geographic distance suggesting that parasite populations are fragmented. Population structure was also identified between villages within the Malala area, with the haplotypes of one parasite population clustering with the neighbouring catchment of Mugil. Conclusion The observed population genetics of P. falciparum in this region is likely to be a consequence of the high transmission intensity combined with the isolation of human and vector populations, especially those located inland and migration of parasites via human movement into coastal populations. The variable genetic diversity and population structure of P. falciparum has important implications for malaria control strategies and warrants further fine scale sampling throughout Papua New Guinea.

Abstract Background Malaria places a significant burden on the limited resources of many low income countries. Knowing more about why and where people seek treatment will enable policy makers to better allocate the limited resources. This study aims to better understand what influences treatment-seeking behaviour for malaria in one such low-income country context, Papua New Guinea (PNG). Methods Two culturally, linguistically and demographically different regions in PNG were selected as study sites. A cross sectional household survey was undertaken in both sites resulting in the collection of data on 928 individuals who reported suffering from malaria in the previous four weeks. A probit model was then used to identify the factors determining whether or not people sought treatment for presumptive malaria. Multinomial logit models also assisted in identifying the factors that determined where people sought treatments. Results Results in this study build upon findings from other studies. For example, while distance in PNG has previously been seen as the primary factor in influencing whether any sort of treatment will be sought, in this study cultural influences and whether it was the first, second or even third treatment for a particular episode of malaria were also important. In addition, although formal health care facilities were the most popular treatment sources, it was also found that traditional healers were a common choice. In turn, the reasons why participants chose a particular type of treatment differed according to the whether they were seeking an initial or subsequent treatments. Conclusions Simply bringing health services closer to where people live may not always result in a greater use of formal health care facilities. Policy makers in PNG need to consider within-country variation in treatment-seeking behaviour, the important role of traditional healers and also ensure that the community fully understands the potential implications of not seeking treatment for illnesses such as malaria at a formal health care facility.

Abstract Ethylene glycol (EG) is metabolized into glycolate and oxalate and may cause metabolic acidemia, neurotoxicity, acute kidney injury (AKI), and death. Historically, treatment of EG toxicity included supportive care, correction of acid–base disturbances and antidotes (ethanol or fomepizole), and extracorporeal treatments (ECTRs), such as hemodialysis. With the wider availability of fomepizole, the indications for ECTRs in EG poisoning are debated. We conducted systematic reviews of the literature following published EXTRIP methods to determine the utility of ECTRs in the management of EG toxicity. The quality of the evidence and the strength of recommendations, either strong (“we recommend”) or weak/conditional (“we suggest”), were graded according to the GRADE approach. A total of 226 articles met inclusion criteria. EG was assessed as dialyzable by intermittent hemodialysis (level of evidence = B) as was glycolate (Level of evidence = C). Clinical data were available for analysis on 446 patients, in whom overall mortality was 18.7%. In the subgroup of patients with a glycolate concentration ≤ 12 mmol/L (or anion gap ≤ 28 mmol/L), mortality was 3.6%; in this subgroup, outcomes in patients receiving ECTR were not better than in those who did not receive ECTR. The EXTRIP workgroup made the following recommendations for the use of ECTR in addition to supportive care over supportive care alone in the management of EG poisoning (very low quality of evidence for all recommendations): i) Suggest ECTR if fomepizole is used and EG concentration > 50 mmol/L OR osmol gap > 50; or ii) Recommend ECTR if ethanol is used and EG concentration > 50 mmol/L OR osmol gap > 50; or iii) Recommend ECTR if glycolate concentration is > 12 mmol/L or anion gap > 27 mmol/L; or iv) Suggest ECTR if glycolate concentration 8–12 mmol/L or anion gap 23–27 mmol/L; or v) Recommend ECTR if there are severe clinical features (coma, seizures, or AKI). In most settings, the workgroup recommends using intermittent hemodialysis over other ECTRs. If intermittent hemodialysis is not available, CKRT is recommended over other types of ECTR. Cessation of ECTR is recommended once the anion gap is

Abstract 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.

Abstract Background Timely presentation and diagnosis of cervical cancer (CC) are crucial to decrease its mortality especially in low- and middle-income countries like Palestine. This study aimed to evaluate the knowledge of Palestinian women about CC warning signs and determine the factors associated with good knowledge. Methods This was a national cross-sectional study conducted between July 2019 and March 2020 in Palestine. Stratified convenience sampling was used to recruit adult women from hospitals, primary healthcare centers, and public spaces of 11 governorates. A translated-into-Arabic version of the validated CC awareness measure (CeCAM) was used to assess women’s knowledge of 12 CC warning signs. Results Of 8086 approached, 7223 participants completed the CeCAM (response rate = 89.3%). A total of 7058 questionnaires were included in the analysis: 2655 from the Gaza Strip and 4403 from the West Bank and Jerusalem (WBJ). The median age [interquartile range] for all participants was 34.0 [24.0, 42.0] years. Participants recruited from the WBJ were older, getting higher monthly income, and having more chronic diseases than those recruited from the Gaza Strip. The most frequently identified warning sign was ‘vaginal bleeding after menopause’ (n = 5028, 71.2%) followed by ‘extreme generalized fatigue’ (n = 4601, 65.2%) and ‘unexplained weight loss’ (n = 4578, 64.9%). Only 1934 participants (27.4%) demonstrated good knowledge of CC warning signs. Participants from the Gaza Strip were slightly more likely than participants from the WBJ to have a good level of knowledge. Factors associated with having good knowledge included having a bachelor or postgraduate degree, being married, divorced, or widowed as well as knowing someone with cancer. Conclusion The overall awareness of CC warning signs was low. Educational interventions are needed to increase Palestinian women’s awareness of CC warning signs.

Abstract 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 (β = 0.015; 95% CI = 0.01–0.03), whereas P. vivax infection was associated with elevation (β = − 0.26; 95% CI = − 0.38 to − 3.04), precipitation during the 3 driest months of the year (β = 0.01; 95% CI = − 0.01–0.02) and slope (β = 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.

Abstract Background Hemodialysis patients with COVID-19 have been reported to be at higher risk for death than the general population. Several prognostic factors have been identified in the studies from Asian, European or American countries. This is the first national Lebanese study assessing the factors associated with SARS-CoV-2 mortality in hemodialysis patients. Methods This is an observational study that included all chronic hemodialysis patients in Lebanon who were tested positive for SARS-CoV-2 from 31st March to 1st November 2020. Data on demographics, comorbidities, admission to hospital and outcome were collected retrospectively from the patients’ medical records. A binary logistic regression analysis was performed to assess risk factors for mortality. Results A total of 231 patients were included. Mean age was 61.46 ± 13.99 years with a sex ratio of 128 males to 103 females. Around half of the patients were diabetics, 79.2% presented with fever. A total of 115 patients were admitted to the hospital, 59% of them within the first day of diagnosis. Hypoxia was the major reason for hospitalization. Death rate was 23.8% after a median duration of 6 (IQR, 2 to 10) days. Adjusted regression analysis showed a higher risk for death among older patients (odds ratio = 1.038; 95% confidence interval: 1.013, 1.065), patients with heart failure (odds ratio = 4.42; 95% confidence interval: 2.06, 9.49), coronary artery disease (odds ratio = 3.27; 95% confidence interval: 1.69, 6.30), multimorbidities (odds ratio = 1.593; 95% confidence interval: 1.247, 2.036), fever (odds ratio = 6.66; 95% confidence interval: 1.94, 27.81), CRP above 100 mg/L (odds ratio = 4.76; 95% confidence interval: 1.48, 15.30), and pneumonia (odds ratio = 19.18; 95% confidence interval: 6.47, 56.83). Conclusions This national study identified older age, coronary artery disease, heart failure, multimorbidities, fever and pneumonia as risk factors for death in patients with COVID-19 on chronic hemodialysis. The death rate was comparable to other countries and estimated at 23.8%.

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1 ).

Abstract 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 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.

Abstract Introduction As malaria transmission declines, understanding the differential impact of intensified control on Plasmodium falciparum relative to Plasmodium vivax and identifying key drivers of ongoing transmission is essential to guide future interventions. Methods Three longitudinal child cohorts were conducted in Papua New Guinea before (2006/2007), during (2008) and after scale-up of control interventions (2013). In each cohort, children aged 1–5 years were actively monitored for infection and illness. Incidence of malaria episodes, molecular force of blood-stage infections (molFOB) and population-averaged prevalence of infections were compared across the cohorts to investigate the impact of intensified control in young children and the key risk factors for malaria infection and illness in 2013. Results Between 2006 and 2008, P. falciparum infection prevalence, molFOB, and clinical malaria episodes reduced by 47%, 59% and 69%, respectively, and a further 49%, 29% and 75% from 2008 to 2013 (prevalence 41.6% to 22.1% to 11.2%; molFOB: 3.4 to 1.4 to 1.0 clones/child/year; clinical episodes incidence rate (IR) 2.6 to 0.8 to IR 0.2 episodes/child/year). P. vivax clinical episodes declined at rates comparable to P. falciparum between 2006, 2008 and 2013 (IR 2.5 to 1.1 to 0.2), while P. vivax molFOB (2006, 9.8; 2008, 12.1) and prevalence (2006, 59.6%; 2008, 65.0%) remained high in 2008. However, in 2013, P. vivax molFOB (1.2) and prevalence (19.7%) had also substantially declined. In 2013, 89% of P. falciparum and 93% of P. vivax infections were asymptomatic, 62% and 47%, respectively, were sub-microscopic. Area of residence was the major determinant of malaria infection and illness. Conclusion Intensified vector control and routine case management had a differential impact on rates of P. falciparum and P. vivax infections but not clinical malaria episodes in young children. This suggests comparable reductions in new mosquito-derived infections but a delayed impact on P. vivax relapsing infections due to a previously acquired reservoir of hypnozoites. This demonstrates the need to strengthen implementation of P. vivax radical cure to maximise impact of control in co-endemic areas. The high heterogeneity of malaria in 2013 highlights the importance of surveillance and targeted interventions to accelerate towards elimination.

Abstract 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.

Abstract Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.

Abstract Background Gene Ontology (GO) is a useful resource of controlled vocabulary that provides information about annotated genes. Based on such resource, finding the biological function is useful for biologists to come up with different hypotheses and help further investigations of an experiment. The biological function for desired genes and gene associations is picked up from a randomly chosen list or through the analysis of differential gene expression. Many tools have been developed to utilize GO knowledge and cluster genes according to relevant biological functions. The retrieved GO terms include both specific and non-specific terms, which is not user-friendly in terms of data analysis. Thus one approach is still missing, which allows navigating through different levels of GO hierarchy manually. Result We developed a tool, GOTrapper, which allows moving up or down to the very bottom of the GO hierarchy. This is performed manually by the user, based on an assigned threshold. This tool grabs the shared terms by the desired set of input genes of Homo sapiens. Here, two inputs are possible. “Within” is to find associated terms within one gene list, and “Between” is to find associated terms between two lists. The tool also provides the option to return the terms with the pre-selected evidence codes. Conclusion GOTrapper is a user-friendly Java tool that helps the user move up and down the ontology tree, which leads to new hypotheses and devising new association of the input genes. It also allows returning terms of associated genes based on selected evidence codes. This tool can be accessed and is freely available at https://github.com/BioGeneTools/GOTrapper.

Abstract 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.

Abstract 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

Abstract 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.

Abstract Background Plasmodium falciparum in pregnancy results in substantial poor health outcomes for both mother and child, particularly in young, primigravid mothers who are at greatest risk of placental malaria (PM) infection. Complications of PM include maternal anaemia, low birth weight and preterm delivery, which contribute to maternal and infant morbidity and mortality in coastal Papua New Guinea (PNG). Methods Placental biopsies were examined from 1451 pregnant women who were enrolled in a malaria prevention study at 14–26 weeks gestation. Clinical and demographic information were collected at first antenatal clinic visits and women were followed until delivery. Placental biopsies were collected and examined for PM using histology. The presence of infected erythrocytes and/or the malaria pigment in monocytes or fibrin was used to determine the type of placental infection. Results Of 1451 placentas examined, PM infection was detected in 269 (18.5%), of which 54 (3.7%) were acute, 55 (3.8%) chronic, and 160 (11.0%) were past infections. Risk factors for PM included residing in rural areas (adjusted odds ratio (AOR) 3.65, 95% CI 1.76–7.51; p ≤ 0.001), being primigravid (AOR 2.45, 95% CI 1.26–4.77; p = 0.008) and having symptomatic malaria during pregnancy (AOR 2.05, 95% CI 1.16–3.62; p = 0.013). After adjustment for covariates, compared to uninfected women, acute infections (AOR 1.97, 95% CI 0.98–3.95; p = 0.056) were associated with low birth weight babies, whereas chronic infections were associated with preterm delivery (AOR 3.92, 95% CI 1.64–9.38; p = 0.002) and anaemia (AOR 2.22, 95% CI 1.02–4.84; p = 0.045). Conclusions Among pregnant PNG women receiving at least one dose of intermittent preventive treatment in pregnancy and using insecticide-treated bed nets, active PM infections were associated with adverse outcomes. Improved malaria prevention is required to optimize pregnancy outcomes.

Abstract Background Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development. Methods ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1–3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up. Results Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33–1.66, P = 0.008–0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65–0.67, P = 0.039–0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax. Conclusions This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.

Abstract 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 αβ 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.

Abstract Background Papua New Guinea (PNG) is a diverse and culturally-rich country with severe infrastructural and health problems. Tuberculosis (TB) is widespread, and the number of cases with drug resistance is rising. Treatment adherence is known to be important for both effective treatment and limiting the emergence of drug resistance. The aim of this study was to construct a matrix of the factors that act as facilitators or barriers to TB treatment adherence in a remote region of PNG. Methods The study was based in the Balimo region of the Western Province. People known to have undergone TB treatment, as well as staff involved in managing people with TB, were asked to participate in an in-depth interview about their experiences. Purposive sampling was used to identify a diverse range of participants, from different geographic locations, social backgrounds, and with successful and unsuccessful treatment outcomes. The interview data was analysed based on grounded theory methodology. Results The study identified a range of factors that influence TB treatment adherence, with these being classified as personal, systems, and sociocultural. These factors are presented along with suggested recommendations for adaptations to DOTS-based treatment in this region. Barriers included the challenges associated with travel to treatment sites, and the difficulties of undertaking treatment alongside the daily need to maintain subsistence food production. However, facilitators were also identified, including the positive influence of religious beliefs, and high confidence in the ability of DOTS-based treatment to cure TB. Conclusions Documenting the wide range of factors that influence treatment adherence in a severely affected remote population will assist in improving TB control. These results provide impetus for further community-based efforts aimed at improving access to TB diagnosis and treatment, and maintaining successful treatment outcomes in the face of emerging drug resistance.

Abstract Background Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. The F protein cleavage site sequence is a well-characterized determinant of NDV pathogenicity in chickens. In this study, the sequences of fusion protein (F) gene of three Newcastle disease virus (NDV) strains isolated from outbreak in chickens in the Al-Sharkia province of Egypt in 2006 were determined. Findings The viral genomic RNAs were extracted from the infective allantoic fluid and F gene is amplified using primer sets designed from the available sequences of NDV strains from GenBank. The pathogenicity of NDV strains was determined by three internationally recognized tests mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index. The phylogenetic analysis showed that the Egypt isolates are closely related with the genotype II of class II NDV strains. Conclusions The sequences of the F genes of the 2006 Egypt isolates are closely related to that of the 2005 Egypt isolate from the same province suggesting that these strains are probably circulating in the vaccinated bird population in Egypt until development of an outbreak.

Abstract Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.

Abstract Background Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world. All APMVs, except avian metapneumovirus, are classified in the genus Avulavirus of the family Paramyxoviridae. At present, the APMVs of genus Avulavirus are divided into nine serological types (APMV 1–9). Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. Very little is known about the molecular characteristics and pathogenicity of APMV 2–9. Results As a first step towards understanding the molecular genetics and pathogenicity of APMV-4, we have sequenced the complete genome of APMV-4 strain duck/Hong Kong/D3/75 and determined its pathogenicity in embryonated chicken eggs. The genome of APMV-4 is 15,054 nucleotides (nt) in length, which is consistent with the "rule of six". The genome contains six non-overlapping genes in the order 3'-N-P/V-M-F-HN-L-5'. The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 9 to 42 nt. The genome contains a 55 nt leader region at 3' end. The 5' trailer region is 17 nt, which is the shortest in the family Paramyxoviridae. Analysis of mRNAs transcribed from the P gene showed that 35% of the transcripts were edited by insertion of one non-templated G residue at an editing site leading to production of V mRNAs. No message was detected that contained insertion of two non-templated G residues, indicating that the W mRNAs are inefficiently produced in APMV-4 infected cells. The cleavage site of the F protein (DIPQR↓F) does not conform to the preferred cleavage site of the ubiquitous intracellular protease furin. However, exogenous proteases were not required for the growth of APMV-4 in cell culture, indicating that the cleavage does not depend on a furin site. Conclusion Phylogenic analysis of the nucleotide sequences of viruses of all five genera of the family Paramyxoviridae showed that APMV-4 is more closely related to the APMVs than to other paramyxoviruses, reinforcing the classification of all APMVs in the genus Avulavirus of the family Paramyxoviridae.

Abstract Background The prevalence of type 2 diabetes in Oman is high and appears to be rising. Rising rates of diabetes and associated risk factors have been observed in populations undergoing epidemiological transition and urbanization. A previous study in Oman indicated that urban-dwellers were not significantly more likely to have diabetes. This study was undertaken to determine if a more accurate urban and rural categorization would reveal different findings. Methods This study included 7179 individuals aged 20 years or above who participated in a cross-sectional interviewer-administered survey in Oman including blood and anthropomorphic tests. Multiple logistic regression analyses were conducted to analyze the factors associated with diabetes, first in the whole population and then stratified according to region. Results The prevalence of diabetes (fasting blood glucose ≥ 7 mmol/l) in the capital region of Muscat was 17.7% compared to 10.5% in rural areas. The prevalence of self-reported diabetes was 4.3%. Urban residence was significantly associated with diabetes (adjusted odds ratio (OR) = 1.7, 95% confidence interval (CI): 1.4–2.1), as was age (OR = 1.2, 95% CI: 1.1- 1.2), obesity (abnormal waist circumference) (OR = 1.8, 95% CI: 1.5–2.1), and systolic blood pressure (SBP) 120–139 (OR = 1.4, 95% CI:1.04–1.8), SBP 140–159 (OR = 1.9, 95% CI: 1.4–2.6), SBP ≥ 160 (OR = 1.7, 95% CI: 1.2–2.5). Stratified analyses revealed higher education was associated with reduced likelihood of diabetes in rural areas (OR = 0.6, 95% CI: 0.4–0.9). Conclusion A high prevalence of diabetes, obesity, hypertension and high cholesterol exist in the Omani population, particularly among urban-dwellers and older individuals. It is vital to continue monitoring chronic disease in Oman and to direct public health policy towards preventing an epidemic.