Fast4DReg - fast registration of 4D microscopy datasets.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

GIANI - open-source software for automated analysis of 3D microscopy images.

The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.

Understanding Fijian health system challenges in the prevention of mother-to-child transmission of HIV services in the three tertiary hospitals in Fiji.

Mother-to-child transmission is the most common route of human immunodeficiency virus transmission in children, which could be prevented with proper treatment and access to care. Health system challenges can impede the effectiveness of prevention of mother-to-child transmission (PMTCT) programmes. We aim to understand the health system challenges to the provision of PMTCT services for pregnant women in three tertiary hospitals in Fiji. Data were collected using in-depth interviews in the three tertiary hospitals and associated health clinics in Fiji between April-May 2013 and February-March 2014. Ethical approvals were obtained. A total of 58 healthcare providers were interviewed including doctors (n = 12), midwives (n = 19), nurses (n = 14), laboratory technicians (n = 5) and counsellors (n = 8). The data were analysed using thematic analysis. We found that the healthcare workers faced a wide range of health system challenges including institutional and human resource challenges. Staff shortage, limited PMTCT training and shortage of supplies were barriers to the quality of PMTCT services. Our findings would be useful in developing strategies to overcome barriers as it would be imperative in improving the quality of PMTCT service provision in Fiji and other similar settings.

Energy, nutrient and overall healthiness of processed packaged foods in Fiji, a comparison between 2018 and 2020.

In Fiji, packaged foods are becoming increasingly available. However, it is unknown if nutrition composition of these foods has changed. This study aims to assess changes in energy, nutrient content and healthiness of packaged foods by comparing data from five major supermarkets in Fiji in 2018 and 2020. Foods were categorised into 14 groups; nutrient composition information was extracted and healthiness assessed using Health Star Rating (HSR). Descriptive statistics and a separate matched products analysis was conducted summarising differences in nutrient content and HSR. There was limited evidence of change in the nutrient content of included products however, there was a small reduction in mean saturated fat in the snack food category (-1.0 g/100 g, 95% CI -1.6 to -0.4 g/100 g). The proportion of products considered healthy based on HSR, increased in the convenience foods category (28.4%, 95% CI 8.3 to 48.5) and decreased in non-alcoholic beverages (-35.2%, 95% CI -43.6 to -26.9). The mean HSR score increased in the fruit and vegetables category (0.1 (95% CI 0.1, 0.2)) and decreased for non-alcoholic beverages (-1.1 (-1.3, -0.9)) and the sauces, dressings, spreads, and dips category (-0.3 (-0.3, -0.2)). Strengthened monitoring of the food supply is needed to improve the healthiness of foods available.

Smile dynamics: A comparative study of Fijians and NZ Europeans.

BACKGROUND: The Fijian 'Bula Smile' is often described as the world's friendliest; however, its description remains anecdotal. OBJECTIVE: This study aimed to describe and compare the dynamics of Fijians' smiles with those of New Zealand Europeans. METHODS: An observational study was conducted on two ethnic groups, Fijians (FJ; N = 23) and New Zealand Europeans (NZ; N = 23), age- and gender-matched. All participants were asked to watch amusing videos, and their reactions were video recorded. The videos were analysed by software to assess the frequency, duration, intensity and genuineness of smiling episodes. Based on the Facial Action Coding System, Action Unit 6 (AU6-cheek raiser), Action Unit 12 (AU12-lip corner puller) and Action Unit 25 (AU25-lips apart) were assessed. Data were analysed by generalised linear models after adjusting for personality traits. RESULTS: Fijians smiled longer than New Zealand Europeans (+19.9%; p = .027). Mean intensity of AU6 (+1.0; 95%CIs = 0.6-1.5; p < .001), AU12 (+0.5; 95%CIs = 0.1-0.9; p = .008) and AU25 (+22.3%; 95%CIs = 7.3%-37.3%; p = .005) were significantly higher in FJ group than the NZ group. CONCLUSION: Smiling features of Fijians and New Zealanders showed objective differences, the most distinctive being a higher activation of the Duchenne's marker (AU6) in the Fijian group, which is regarded as a sign of smile genuineness.

Rapid SARS-CoV-2 Seroprevalence Survey in Central and Western Divisions of Fiji, 2021.

During November-December 2021, we performed a SARS-CoV-2 seroprevalence survey in Central and Western Divisions of Fiji. A total of 539 participants 8-70 years of age were 95.5% (95% CI 93.4%-97.1%) seropositive, indicating high community levels of immunity. Seroprevalence studies can inform public health responses to emerging SARS-CoV-2 variants.

Zebrafish dnm1a gene plays a role in the formation of axons and synapses in the nervous tissue.

Classical dynamins (DNMs) are GTPase proteins engaged in endocytosis, a fundamental process for cargo internalization from the plasma membrane. In mammals, three DNM genes are present with different expression patterns. DNM1 is expressed at high levels in neurons, where it takes place in the recycling of synaptic vesicles; DNM2 is ubiquitously expressed, while DNM3 is found in the brain and in the testis. Due to the conservation of genes in comparison to mammals, we took advantage of a zebrafish model for functional characterization of dnm1a, ortholog of mammalian DNM1. Our data strongly demonstrated that dnm1a has a nervous tissue-specific expression pattern and plays a role in the formation of both axon and synapse. This is the first in vivo study that collects evidence about the effects of dnm1a loss of function in zebrafish, thus providing a new excellent model to be used in different scientific fields.

The challenges in protecting public health interests in multisectoral governance in the context of small island developing states: the case of tobacco control in Fiji and Vanuatu.

BACKGROUND: The commercial determinants of health (CDoH) drive the rise of NCDs globally, and their regulation requires multisectoral governance. Despite existing recommendations to strengthen institutional structures, protecting public health interests can be challenging amidst industry interference and conflicting policy priorities, particularly in low and middle-income countries (LMICs) where the need for rapid economic development is pronounced. Small island developing states (SIDS) face even more challenges in regulating CDoH because their unique socioeconomic, political, and geographic vulnerabilities may weaken institutional conditions that could aid health sector actors in protecting health interests. This study aims to explore the institutional conditions that shape health sector actors' capability to protect public health interests in tobacco governance in Fiji and Vanuatu. METHODS: We employed a qualitative, exploratory case study design. We applied the administrative process theory to inform data collection and analysis. Seventy interviews were completed in Fiji and Vanuatu from 2018 to 2019. RESULTS: The findings show that the protection of health interests in tobacco governance were not supported by the institutional conditions in Fiji and Vanuatu. While the policy processes formally ensured a level playing field between actors, policies were often developed through informal mechanisms, and the safeguards to protect public interests from vested private interests were not implemented adequately. SIDS vulnerabilities and weak regulation of political parties contributed to the politicisation of government in both states, resulting in high-level government officials' questionable commitment to protect public health interests. The system of checks and balances usually embedded into democratic governments appeared to be muted, and policymakers had limited bureaucratic autonomy to elevate health interests in multisectoral policymaking amidst high-level government officials' frequent rotation. Finally, capacity constraints aggravated by SIDS vulnerabilities negatively impacted health sector actors' capability to analyse policy alternatives. CONCLUSIONS: Health sector actors in Fiji and Vanuatu were not supported by institutional conditions that could help them protect public health interests in multisectoral governance to regulate CDoH originating from the tobacco industry. Institutional conditions in these states were shaped by SIDS vulnerabilities but could be improved by targeted capacity building, governance and political system strengthening.

Predictors of Knowledge, Attitude, and Practice (KAP) Towards Family Planning (FP) Among Pregnant Women in Fiji.

OBJECTIVE: This study aimed to determine the predictors of Knowledge, Attitude and Practice (KAP) towards Family Planning (FP) among pregnant Fijian women. METHODS: A cross-sectional study was conducted over two months in 2019 with adult pregnant women attending the Antenatal Clinic (ANC) at Ba Mission Hospital (BMH), Fiji. Data was collected using a self-administrated questionnaire. Statistical analysis included correlation tests and regression analysis in determining predictors of KAP. RESULTS: 240 pregnant women participated in this study with a mean age of 26.02 (± SD = 4.13). The results showed a moderate level of knowledge (mean 14.95, SD ± 3.15), positive attitude (mean 20.56, SD ± 5.68), and good practice (mean 4.97, SD ± 1.73). Linear regression identified that women with more than seven children had a knowledge score of 3.65, lower than null parity (t value = -2.577, p = 0.011). Women aged 20 to 24 had a 6.47 lower attitude score than women aged 18 to 19 (t value = -2.142, p = 0.033). Women in defacto relationships had a 2.12 lower attitude score compared to the married category (t value = -2.128, p = 0.034). Fijian women of Indian descent had a 1.98 lower attitude score than the I Taukei women (t value = -2.639, p = 0.009). Women aged 30-34 had 2.41 lower practice scores than those aged 18-19 (t value = -2.462, p = 0.015). CONCLUSION: This study found a medium knowledge of FP among pregnant women. These findings support a recommendation for further research to implement effective strategies.

Extracting multiple surfaces from 3D microscopy images in complex biological tissues with the Zellige software tool.

BACKGROUND: Efficient tools allowing the extraction of 2D surfaces from 3D-microscopy data are essential for studies aiming to decipher the complex cellular choreography through which epithelium morphogenesis takes place during development. Most existing methods allow for the extraction of a single and smooth manifold of sufficiently high signal intensity and contrast, and usually fail when the surface of interest has a rough topography or when its localization is hampered by other surrounding structures of higher contrast. Multiple surface segmentation entails laborious manual annotations of the various surfaces separately. RESULTS: As automating this task is critical in studies involving tissue-tissue or tissue-matrix interaction, we developed the Zellige software, which allows the extraction of a non-prescribed number of surfaces of varying inclination, contrast, and texture from a 3D image. The tool requires the adjustment of a small set of control parameters, for which we provide an intuitive interface implemented as a Fiji plugin. CONCLUSIONS: As a proof of principle of the versatility of Zellige, we demonstrate its performance and robustness on synthetic images and on four different types of biological samples, covering a wide range of biological contexts.

Determination of trace cobalt ions in bottled drinking water samples from Fiji Island by spray-assisted fine droplet formation-liquid phase microextraction based on simultaneous complexation and extraction before flame atomic absorption spectrometer measurement.

In this study, a green, simple and effective preconcentration method named as spray-assisted fine droplet formation-liquid phase microextraction (SAFDF-LPME) before the flame atomic absorption spectrophotometry (FAAS) measurement for cobalt determination was developed. The method reduces the external dispersive solvent usage by using a simple spraying apparatus to obtain fine droplets of the extraction solvent. SAFDF-LPME method also consists of simultaneous complexation and extraction which indicates the environmental benevolence of the developed method. This method minimized the relative errors with high repeatability and accuracy by reducing the experimental steps. The influential parameters such as buffer type, buffer solution volume, extraction solvent/ligand solution volume (spraying cycle), and mixing period were systematically optimized by the univariate optimization procedure. With the optimum parameters applied, the detection power of the FAAS system was enhanced to about 110-folds with respect to 2.2 ng mL-1 detection limit calculated for the proposed method. Bottled drinking water samples from Fiji Islands were used to demonstrate the applicability of the developed method for the accurate determination of trace cobalt in real sample matrices. Percent recovery results obtained between 95.5 and 88.5% showed the suitability of the developed method in the determination of cobalt at trace levels even in complex sample matrices.

Socio-ecological challenges and food security in the 'salad bowl' of Fiji, Sigatoka Valley.

This article examines food security in the Sigatoka Valley, one of the most productive food regions in Fiji, in the context of recent socio-ecological challenges through a case study of Narewa village. Data were collected using semi-structured interviews (n = 25), a fixed question food insecurity experience survey (n = 25), and a free listing exercise about preferred and consumed foods (n = 24). Results revealed that while most households had access to sufficient food, the increased frequency and intensity of droughts, tropical cyclones, and flooding caused almost half to worry about meeting their future food needs. To date, a culture of sharing within the village has helped most households access food but this will likely be inadequate to meet future needs as climate change is projected to impact food production. Given that the foundation of food production in Narewa, like other villages in the valley, relies on the long-term viability of agricultural systems, better focus needs to be placed on the natural resources that form the backbone of these systems such as water availability, soil health, and slope stability and their resilience to anthropogenic and natural stressors. Efforts that focus on protecting and enhancing local ecosystems in light of expected future climate change, combined with greater attention on food storage and the use of resilient crops, and enhancing social cohesion and sharing networks are needed to avoid breaching tipping points in the food system.

Climate change and coastal resiliency of Suva, Fiji: a holistic approach for measuring climate risk using the climate and ocean risk vulnerability index (CORVI).

Coastal cities are under severe threat from the impacts of climate change, such as sea-level rise, extreme weather events, coastal inundation, and ecosystem degradation. It is well known that the ocean, and in particular coastal environments, have been changing at an unprecedented rate, which poses increasing risks to people in small island developing states, such as Fiji. The Greater Suva Urban Area, the capital and largest metropolitan area of Fiji, is expected to be largely impacted by climate-related risks to its socio-economic, cultural, and political positions. In the face of these threats, creating a resilient city that can withstand and adapt to the impacts of climate change and promote sustainable development should be guided by a holistic approach, encompassing stakeholders from the government, the private sector, civil society organizations, and international institutions. This study assesses the risk profile of Suva city using an innovative risk information tool, the climate and ocean risk vulnerability index (CORVI), which applies structured expert judgment to quantify climate-related risks in data-sparse environments. Through comparative quantification of diverse risk factors and narrative analysis, this study identifies three priority areas for Suva's future climate-resilient actions: development of climate risk-informed urban planning, harmonized urban development and natural restoration, and enhancing the climate resilience to the tourism sector. Supplementary Information: The online version contains supplementary material available at 10.1007/s11027-022-10043-4.

Poji: a Fiji-based tool for analysis of podosomes and associated proteins.

Podosomes are actin-based adhesion and invasion structures in a variety of cell types, with podosome-forming cells displaying up to several hundreds of these structures. Podosome number, distribution and composition can be affected by experimental treatments or during regular turnover, necessitating a tool that is able to detect even subtle differences in podosomal properties. Here, we present a Fiji-based macro code termed 'Poji' ('podosome analysis by Fiji'), which serves as an easy-to-use tool to characterize a variety of cellular and podosomal parameters, including area, fluorescence intensity, relative enrichment of associated proteins and radial podosome intensity profiles. This tool should be useful to gain more detailed insight into the regulation, architecture and functions of podosomes. Moreover, we show that Poji is easily adaptable for the analysis of invadopodia and associated extracellular matrix degradation, and likely also of other micron-size punctate structures. This article describes the workflow of the Poji macro, presents several examples of its applications, and also points out limitations, as well as respective solutions, and adaptable features to streamline the analysis.This article has an associated First Person interview with the first author of the paper.

Quantification of Lipid Area within Thermogenic Mouse Perivascular Adipose Tissue Using Standardized Image Analysis in FIJI.

Quantification of adipocyte size and number is routinely performed for white adipose tissues using existing image analysis software. However, thermogenic adipose tissue has multilocular adipocytes, making it difficult to distinguish adipocyte cell borders and to analyze lipid proportion using existing methods. We developed a simple, standardized method to quantify lipid content of mouse thermogenic adipose tissue. This method, using FIJI analysis of hematoxylin/eosin stained sections, was highly objective and highly reproducible, with ∼99% inter-rater reliability. The method was compared to direct lipid staining of adipose tissue, with comparable results. We used our method to analyze perivascular adipose tissue (PVAT) from C57BL/6 mice on a normal chow diet, compared to calorie restriction or a high fat diet, where lipid storage phenotypes are known. Results indicate that lipid content can be estimated within mouse PVAT in a quantitative and reproducible manner, and shows correlation with previously studied molecular and physiological measures.

A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges.

Fluorescence lifetime imaging (FLIM) allows the quantification of sub-cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time-domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre-of-mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least-square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time-domain FLIM data set.

Incorporating a gender lens into nutrition and health-related policies in Fiji: analysis of policies and stakeholder perspectives.

BACKGROUND: Gender equality, zero hunger and healthy lives and well-being for all, are three of the Sustainable Development Goals (SDGs) that underpin Fiji's National Development Plan. Work towards each of these goals contributes to the reduction of non-communicable diseases (NCDs). There are gender differences in NCD burden in Fiji. It is, however, unclear whether a gender lens could be more effectively included in nutrition and health-related policies. METHODS: This study consisted of three components: (i) a policy content analysis of gender inclusion in nutrition and health-related policies (n = 11); (ii) policy analysis using the WHO Gender Analysis tool to identify opportunities for strengthening future policy; and (iii) informant interviews (n = 18), to understand perceptions of the prospects for gender considerations in future policies. RESULTS: Gender equality was a goal in seven policies (64%); however, most focused on women of reproductive age. One of the policies was ranked as gender responsive. Main themes from key informant interviews were: 1) a needs-based approach for the focus on specific population groups in policies; 2) gender-related roles and responsibilities around nutrition and health; 3) what is considered "equitable" when it comes to gender, nutrition, and health; 4) current considerations of gender in policies and ideas for further gender inclusion; and 5) barriers and enablers to the inclusion of gender considerations in policies. Informants acknowledged gender differences in the burden of nutrition-related NCDs, yet most did not identify a need for stronger inclusion of gender considerations within policies. CONCLUSIONS: There is considerable scope for greater inclusion of gender in nutrition and health-related policies in Fiji. This could be done by: 1) framing gender considerations in ways that are actionable and inclusive of a range of gender identities; 2) undertaking advocacy through actor networks to highlight the need for gender-responsive nutrition and health-related policies for key stakeholder groups; 3) ensuring that data collected to monitor policy implementation is disaggregated by sex and genders; and 4) promoting equitable participation in nutrition related issues in communities and governance processes. Action on these four areas are likely critical enablers to more gender equitable NCD reduction in Fiji.

Psychometric properties of the Child-OIDP and oral health-related quality of life (OHRQoL) in secondary schools in Suva, Fiji.

BACKGROUND: Oral health-related problems are highly prevalent and, like many other diseases, affect Quality of Life. Although most primary schools in Fiji have supervised school brushing programs and have regular screenings these preventive aspects are missing in secondary schools. OBJECTIVE: To assess the internal consistency reliability, face and content validity of the Child-OIDP questionnaire and determine the oral health-related quality of life in 15-year-olds in Suva, Fiji. METHODS: A cross-sectional prospective study was carried out on 15-year-old children from four secondary schools in Suva, Fiji from 2014 to 2015. All students enrolled in the 10th and 11th year of studies were included. Multi-stage cluster sampling was used to identify the participants and the sample size of 367 was calculated. The Child Oral Impact on Daily Performance (Child-OIDP) self-administered questionnaire was used to collect data and data was analyzed using Epi-Info (3.5.1). RESULTS: A total of 281 students (76.6%) responded, of whom 47.0% experienced at least one impact. Cronbach's alpha for the Child-OIDP frequency items was 0.70 and the corrected item-total correlation ranged from 0.13 to 0.57. The most common performances that were affected were eating (27.8%) and relaxing (12.8%). Performances that were severely and most severely influenced were social contact (23.1%), smiling (16.7%) and relaxing (16.7%). The most common conditions leading to impacts were dental sensitivity (38.4%), dental caries (23.5%) and toothache (21.4%). CONCLUSION: The original version of the Child-OIDP is a reliable index with acceptable internal consistency when used directly in the Fiji setting, however, further studies to validate the tool will be useful. Oral impacts were prevalent, but not severe.

Perception of pregnant women towards early antenatal visit in Fiji: a qualitative study.

BACKGROUND: Antenatal Care (ANC) is an opportunity to provide care to prevent potential maternal and new born mortality and morbidity and reduce new born mortality and morbidity. There has been an increase in the number of women receiving early ANC over the last two decades, however, in many developing regions such as Fiji, women are still delaying initiation of ANC. Therefore, the aim of this study is primarily to explore reasons for delayed initiation of ANC appointments and to explore knowledge and perception of pregnant mothers towards early antenatal appointments in Fiji. METHODS: The study uses a qualitative approach. Data was collected among pregnant women more than 18 years of age after 12 weeks of gestation attending their first ANC clinic at the Ba Mission Hospital (BMH) from February 28 to April 2, 2020. Heterogenous purposeful sampling method was used to select 25 pregnant women for the study. A semi-structured open-ended questionnaire was used for face to face in-depth interviews. Data was analyzed manually using thematic content analysis after verbatim transcription of the interviews. RESULTS: The mean age of the participants was 25.8 ± 5.9 years (age range of 19-40 years). The average gestational age of those making a booking for a consultation was 5.4 ± 1.4 months with a range of 4 to 8 months. The majority of women were multigravida (64%) and multiparous (40%). The main themes that emerged from the study were: i) perception of early ANC booking; ii) perceived barriers of early ANC booking and; iii) enabling factors of early ANC booking. Even though pregnant women have a good knowledge of when to initiate ANC, the practice of early booking was influenced by many other factors. CONCLUSIONS: The results of this study highlight the need to change the current booking system. Efforts are needed to attract the hard-to-reach women through outreach visits and increased communication between health care workers and the community with the use of community resources such as community health workers and traditional birth attendants. The media should be used to create awareness on timing and importance of early ANC visits at a community level.