Diarrhea Case Surveillance in the Enterics for Global Health Shigella Surveillance Study: Epidemiologic Methods.

Background: Shigella is a leading cause of acute watery diarrhea, dysentery, and diarrhea-attributed linear growth faltering, a precursor to stunting and lifelong morbidity. Several promising Shigella vaccines are in development and field efficacy trials will require a consortium of potential vaccine trial sites with up-to-date Shigella diarrhea incidence data. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will employ facility-based enrollment of diarrhea cases aged 6-35 months with 3 months of follow-up to establish incidence rates and document clinical, anthropometric, and financial consequences of Shigella diarrhea at 7 country sites (Mali, Kenya, The Gambia, Malawi, Bangladesh, Pakistan, and Peru). Over a 24-month period between 2022 and 2024, the EFGH study aims to enroll 9800 children (1400 per country site) between 6 and 35 months of age who present to local health facilities with diarrhea. Shigella species (spp.) will be identified and serotyped from rectal swabs by conventional microbiologic methods and quantitative polymerase chain reaction. Shigella spp. isolates will undergo serotyping and antimicrobial susceptibility testing. Incorporating population and healthcare utilization estimates from contemporaneous household sampling in the catchment areas of enrollment facilities, we will estimate Shigella diarrhea incidence rates. Conclusions: This multicountry surveillance network will provide key incidence data needed to design Shigella vaccine trials and strengthen readiness for potential trial implementation. Data collected in EFGH will inform policy makers about the relative importance of this vaccine-preventable disease, accelerating the time to vaccine availability and uptake among children in high-burden settings.

Quantifying the Cost of Shigella Diarrhea in the Enterics for Global Health (EFGH) Shigella Surveillance Study.

Background: Comparative costs of public health interventions provide valuable data for decision making. However, the availability of comprehensive and context-specific costs is often limited. The Enterics for Global Health (EFGH) Shigella surveillance study-a facility-based diarrhea surveillance study across 7 countries-aims to generate evidence on health system and household costs associated with medically attended Shigella diarrhea in children. Methods: EFGH working groups comprising representatives from each country (Bangladesh, Kenya, Malawi, Mali, Pakistan, Peru, and The Gambia) developed the study methods. Over a 24-month surveillance period, facility-based surveys will collect data on resource use for the medical treatment of an estimated 9800 children aged 6-35 months with diarrhea. Through these surveys, we will describe and quantify medical resources used in the treatment of diarrhea (eg, medication, supplies, and provider salaries), nonmedical resources (eg, travel costs to the facility), and the amount of caregiver time lost from work to care for their sick child. To assign costs to each identified resource, we will use a combination of caregiver interviews, national medical price lists, and databases from the World Health Organization and the International Labor Organization. Our primary outcome will be the estimated cost per inpatient and outpatient episode of medically attended Shigella diarrhea treatment across countries, levels of care, and illness severity. We will conduct sensitivity and scenario analysis to determine how unit costs vary across scenarios. Conclusions: Results from this study will contribute to the existing body of literature on diarrhea costing and inform future policy decisions related to investments in preventive strategies for Shigella.

Prevalence and factors related to anaemia in children aged 6-59 months attending a quaternary health facility in Maputo, Mozambique.

Globally, anaemia prevails as a public health issue, being also a concern in Mozambique, where about two-thirds of children 6-59 months of age are affected by this condition. We carried out this study to estimate anaemia prevalence and evaluate structural determinants and haematological parameters association among children aged 6-59 months attending pediatric inpatient and outpatient services in a Quaternary Health Facility in Maputo City Province, Mozambique. We collected data from 637 inpatients or outpatients who attended pediatric consultations at the Maputo Central Hospital. The overall rate of anaemia in children aged 6-59 months was 62.2% (396/637), with 30.9% moderate anaemia (197/637), 23.9% mild anaemia (152/637), and 7.4% severe anaemia (47/637). Among our study participants, critical factors for anaemia were those concerning the age group, child´s caregiver schooling, malaria and size of the liver.

Improving Antibiotic Stewardship for Diarrheal Disease With Probability-Based Electronic Clinical Decision Support: A Randomized Crossover Trial.

Importance: Inappropriate use of antibiotics for diarrheal illness can result in adverse effects and increase in antimicrobial resistance. Objective: To determine whether the diarrheal etiology prediction (DEP) algorithm, which uses patient-specific and location-specific features to estimate the probability that diarrhea etiology is exclusively viral, impacts antibiotic prescriptions in patients with acute diarrhea. Design, Setting, and Participants: A randomized crossover study was conducted to evaluate the DEP incorporated into a smartphone-based electronic clinical decision-support (eCDS) tool. The DEP calculated the probability of viral etiology of diarrhea, based on dynamic patient-specific and location-specific features. Physicians were randomized in the first 4-week study period to the intervention arm (eCDS with the DEP) or control arm (eCDS without the DEP), followed by a 1-week washout period before a subsequent 4-week crossover period. The study was conducted at 3 sites in Bangladesh from November 17, 2021, to January 21, 2021, and at 4 sites in Mali from January 6, 2021, to March 5, 2021. Eligible physicians were those who treated children with diarrhea. Eligible patients were children between ages 2 and 59 months with acute diarrhea and household access to a cell phone for follow-up. Interventions: Use of the eCDS with the DEP (intervention arm) vs use of the eCDS without the DEP (control arm). Main Outcomes and Measures: The primary outcome was the proportion of children prescribed an antibiotic. Results: A total of 30 physician participants and 941 patient participants (57.1% male; median [IQR] age, 12 [8-18] months) were enrolled. There was no evidence of a difference in the proportion of children prescribed antibiotics by physicians using the DEP (risk difference [RD], -4.2%; 95% CI, -10.7% to 1.0%). In a post hoc analysis that accounted for the predicted probability of a viral-only etiology, there was a statistically significant difference in risk of antibiotic prescription between the DEP and control arms (RD, -0.056; 95% CI, -0.128 to -0.01). No known adverse effects of the DEP were detected at 10-day postdischarge. Conclusions and Relevance: Use of a tool that provides an estimate of etiological likelihood did not result in a significant change in overall antibiotic prescriptions. Post hoc analysis suggests that a higher predicted probability of viral etiology was linked to reductions in antibiotic use. Trial Registration: Clinicaltrials.gov Identifier: NCT04602676.

Introduction of an Electronic Clinical Decision Support Tool to Inform Prescribing for Pediatric Diarrhea in Bangladesh and Mali: Do Provider Expectations Predict Experiences?

Nonindicated antibiotics for childhood diarrhea is a major contributor to global antimicrobial resistance. Electronic clinical decision support tools (eCDSTs) may reduce unnecessary antibiotics. This study examined how providers' expectations of an eCDST to predict diarrhea etiology compared with their experiences using the tool. Providers were enrolled from public hospitals in Bangladesh (n = 15) and Mali (n = 15), and surveys were completed at baseline and after using the eCDST. Baseline surveys assessed expectations (utility, ease of use, and threat to autonomy), and post surveys assessed experiences in the same domains. Providers' experiences with ease of use exceeded their baseline expectations, and providers reported less experienced threat to autonomy after use, compared with baseline expectations. Providers' expectations of threat to autonomy significantly predicted their experienced threat to autonomy. Findings suggest that an eCDST to inform antimicrobial prescribing for diarrhea is feasible and acceptable, but training should promote local ownership for sustainability.

Can We Use Routine Data for Strategic Decision Making? A Time Trend Comparison Between Survey and Routine Data in Mali.

BACKGROUND: Countries with scarce resources need timely and high-quality data on coverage of health interventions to make strategic decisions about where to allocate investments in health. Household survey data are generally regarded as "gold standard," high-quality data. This study assessed the comparability of intervention coverage time trends from routine and survey data at national and subnational levels in Mali. METHODS: We compared 3 coverage indicators: contraceptive prevalence rate, institutional delivery, and 3 doses of diphtheria, pertussis, and tetanus (DPT3) vaccine, using 3 Mali Demographic and Health Surveys (DHS 2001, 2006, and 2012-2013) and routine health system data covering 2001-2012. For routine data, we used local health information system (HIS) annual reports and an HIS database. To compare time trends between the data sources, we calculated the percentage point change and 95% confidence interval from 2001-2006 and 2006-2012. We then computed the absolute and relative differences between the 2 data sources for each indicator over time at national and regional levels and assessed their level of significance. RESULTS: The direction and magnitude of the time trends of contraceptive prevalence rate, institutional delivery, and DPT3 vaccine from 2001 to 2012 were similar at the national level between data sources. At the regional level, there were significant differences in the magnitude and direction of time trends for institutional delivery and the DPT3 vaccine; contraceptive prevalence trends were more consistent. Routine data tended to overestimate DPT3 coverage, and underestimate institutional delivery and contraceptive prevalence relative to survey data. CONCLUSION: Routine data in Mali-particularly at the national level-appear to be appropriate for use to inform program planning and prioritization, but routine time trends should be interpreted with caution at the subnational level. For program evaluations, routine data may not be appropriate to draw accurate inferences about program impact.

Effect of mobile application user interface improvements on minimum expected home visit coverage by community health workers in Mali: a randomised controlled trial.

BACKGROUND: Proactive community case management (ProCCM) has shown promise to advance goals of universal health coverage (UHC). ProCCM community health workers (CHWs) face operational challenges when pursuing their goal of visiting every household in their service area at least twice monthly to proactively find sick patients. We developed a software extension (UHC Mode) to an existing CHW mobile application featuring user interface design improvements to support CHWs in planning daily home visits. We evaluated the effect of UHC Mode on minimum expected home visit coverage. METHODS: We conducted a parallel-group, two-arm randomised controlled trial of ProCCM CHWs in two separate regions in Mali. CHWs were randomly assigned to UHC Mode or the standard mobile application (control) with a 1:1 allocation. Randomisation was stratified by health catchment area. CHWs and other programme personnel were not masked to arm allocation. CHWs used their assigned intervention for 4 months. Using a difference-in-differences analysis, we estimated the mean change in minimum expected home visit coverage from preintervention to postintervention between arms. RESULTS: Enrolment occurred in January 2019. Of 199 eligible CHWs randomised to the intervention or control arm, 196 were enrolled and 195 were included in the analysis. Households whose CHW used UHC Mode had 2.41 times higher odds of minimum expected home visit coverage compared with households whose CHW used the control (95% CI 1.68 to 3.47; p<0.0005). Minimum expected home visit coverage in the UHC Mode arm increased 13.6 percentage points (95% CI 8.1 to 19.0) compared with the control arm. CONCLUSION: Our findings suggest UHC Mode is an effective tool that can improve home visit coverage and promote progress towards UHC when implemented in the ProCCM context. User interface design of health information systems that supports health workers' daily practices and meets their requirements can have a positive impact on health worker performance and home visit coverage. TRIAL REGISTRATION NUMBER: NCT04106921.

Women's empowerment, intrahousehold influences, and health system design on modern contraceptive use in rural Mali: a multilevel analysis of cross-sectional survey data.

BACKGROUND: Persistent challenges in meeting reproductive health and family planning goals underscore the value in determining what factors can be leveraged to facilitate modern contraceptive use, especially in poor access settings. In Mali, where only 15% of reproductive-aged women use modern contraception, understanding how women's realities and health system design influence contraceptive use helps to inform strategies to achieve the nation's target of 30% by 2023. METHODS: Using household survey data from the baseline round of a cluster-randomized trial, including precise geolocation data from all households and public sector primary health facilities, we used a multilevel model to assess influences at the individual, household, community, and health system levels on women's modern contraceptive use. In a three-level, mixed-effects logistic regression, we included measures of women's decision-making and mobility, as well as socio-economic sources of empowerment (education, paid labor), intrahousehold influences in the form of a co-residing user, and structural factors related to the health system, including distance to facility. RESULTS: Less than 5% of the 14,032 women of reproductive age in our study used a modern method of contraception at the time of the survey. Women who played any role in decision-making, who had any formal education and participated in any paid labor, were more likely to use modern contraception. Women had three times the odds of using modern contraception if they lived in a household with another woman, typically a co-wife, who also used a modern method. Compared to women closest to a primary health center, those who lived between 2 and 5 km were half as likely to use modern contraception, and those between 5 and 10 were a third as likely. CONCLUSIONS: Despite chronically poor service availability across our entire study area, some women-even pairings of women in single households-transcended barriers to use modern contraception. When planning and implementing strategies to expand access to contraception, policymakers and practitioners should consider women's empowerment, social networks, and health system design. Accessible and effective health systems should reconsider the conventional approach to community-based service delivery, including distance as a barrier only beyond 5 km.

RéSUMé: CONTEXTE: Au Mali, où seulement 15% des femmes en âge de procréer utilisent les contraceptifs modernes, la compréhension des réalités des femmes et de la conception du système de santé aident à éclairer les stratégies pour atteindre l'objectif national de 30% d'ici 2023. MéTHODES: En utilisant les données d'enquête de base d'un essai randomisé en grappes, avec la géolocalisation précise de tous les ménages et centres de santé publiques, nous avons utilisé un modèle à plusieurs niveaux pour évaluer l'influence de l'individu, du ménage, de la communauté et du système de santé sur l'utilisation de la contraception moderne. Nous avons utilisé la régression logistique à effets mixtes pour mesurer l'autonomisation et ses sources socio-économiques (éducation, travail rémunéré), les influences intra-ménages sous forme d'une utilisatrice co-résidante et les facteurs structurels liés au système de santé. RéSULTATS: Moins de 5% des 14 032 femmes en âge de procréer utilisaient la contraception moderne au moment de l'enquête. Les femmes jouant un rôle dans la prise de décision, celles ayant une éducation formelle, un travail rémunéré, étaient plus susceptibles d'utiliser les contraceptifs modernes. Les femmes avaient trois fois plus de chances de faire la contraception moderne si elles vivaient dans un ménage avec une autre femme, généralement une coépouse, qui utilisait une méthode moderne. Comparées aux femmes les plus proches d'un centre de santé, celles qui vivaient entre 2 and 5 kilomètres étaient deux fois moins susceptibles d'utiliser un contraceptif moderne et celles entre 5 and 10 étaient plus susceptibles dans un tiers des cas. CONCLUSIONS: Malgré une faible disponibilité des services dans toute la zone d'étude, certaines femmes­même celles en cohabitation­ont pu surmonter les barrières à l'utilisation des contraceptifs modernes. Lors de la planification et de la mise en œuvre de stratégies pour élargir l'accès à la contraception, les décideurs et les praticiens devraient tenir compte de l'autonomisation des femmes, des réseaux sociaux, et de la conception du système de santé. Les systèmes de santé accessibles et efficaces devraient reconsidérer l'approche conventionnelle de la prestation de services communautaires, en prenant en compte la distance même à moins de 5 kilomètres.

Timing and number of antenatal care contacts in low and middle-income countries: Analysis in the Countdown to 2030 priority countries.

BACKGROUND: The 2016 World Health Organization (WHO) guidelines for antenatal care (ANC) shift the recommended minimum number of ANC contacts from four to eight, specifying the first contact to occur within the first trimester of pregnancy. We quantify the likelihood of meeting this recommendation in 54 Countdown to 2030 priority countries and identify the characteristics of women being left behind. METHODS: Using 54 Demographic and Health Surveys (DHS) and Multiple Indicator Cluster Surveys (MICS) since 2012, we reported the proportion of women with timely ANC initiation and those who received 8-10 contacts by coverage levels of ANC4+ and by Sustainable Development Goal (SDG) regions. We identified demographic, socio-economic and health systems characteristics of timely ANC initiation and achievement of ANC8+. We ran four multiple regression models to quantify the associations between timing of first ANC and the number and content of ANC received. RESULTS: Overall, 49.9% of women with ANC1+ and 44.3% of all women had timely ANC initiation; 11.3% achieved ANC8+ and 11.2% received no ANC. Women with timely ANC initiation had 5.2 (95% confidence interval (CI) = 5.0-5.5) and 4.7 (95% CI = 4.4-5.0) times higher odds of receiving four and eight ANC contacts, respectively (P < 0.001), and were more likely to receive a higher content of ANC than women with delayed ANC initiation. Regionally, women in Central and Southern Asia had the best performance of timely ANC initiation; Latin America and Caribbean had the highest proportion of women achieving ANC8+. Women who did not initiate ANC in the first trimester or did not achieve 8 contacts were generally poor, single women, with low education, living in rural areas, larger households, having short birth intervals, higher parity, and not giving birth in a health facility nor with a skilled attendant. CONCLUSIONS: Timely ANC initiation is likely to be a major driving force towards meeting the 2016 WHO guidelines for a positive pregnancy experience.

Proactive community case management and child survival: protocol for a cluster randomised controlled trial.

INTRODUCTION: Community health workers (CHWs)-shown to improve access to care and reduce maternal, newborn, and child morbidity and mortality-are re-emerging as a key strategy to achieve health-related Sustainable Development Goals (SDGs). However, recent evaluations of national programmes for CHW-led integrated community case management (iCCM) of common childhood illnesses have not found benefits on access to care and child mortality. Developing innovative ways to maximise the potential benefits of iCCM is critical to achieving the SDGs. METHODS AND ANALYSIS: An unblinded, cluster randomised controlled trial in rural Mali aims to test the efficacy of the addition of door-to-door proactive case detection by CHWs compared with a conventional approach to iCCM service delivery in reducing under-five mortality. In the intervention arm, 69 village clusters will have CHWs who conduct daily proactive case-finding home visits and deliver doorstep counsel, care, referral and follow-up. In the control arm, 68 village clusters will have CHWs who provide the same services exclusively out of a fixed community health site. A baseline population census will be conducted of all people living in the study area. All women of reproductive age will be enrolled in the study and surveyed at baseline, 12, 24 and 36 months. The survey includes a life table tracking all live births and deaths occurring prior to enrolment through the 36 months of follow-up in order to measure the primary endpoint: under-five mortality, measured as deaths among children under 5 years of age per 1000 person-years at risk of mortality. ETHICS AND DISSEMINATION: The trial has received ethical approval from the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry, University of Bamako. The results will be disseminated through peer-reviewed publications, national and international conferences and workshops, and media outlets. TRIAL REGISTRATION NUMBER: NCT02694055; Pre-results.

Improving Community Health Worker performance by using a personalised feedback dashboard for supervision: a randomised controlled trial.

BACKGROUND: Countries across sub-Saharan Africa are scaling up Community Health Worker (CHW) programmes, yet there remains little high-quality research assessing strategies for CHW supervision and performance improvement. This randomised controlled trial aimed to determine the effect of a personalised performance dashboard used as a supervision tool on the quantity, speed, and quality of CHW care. METHODS: We conducted a randomised controlled trial in a large health catchment area in peri-urban Mali. One hundred forty-eight CHWs conducting proactive case-finding home visits were randomly allocated to receive individual monthly supervision with or without the CHW Performance Dashboard from January to June 2016. Randomisation was stratified by CHW supervisor, level of CHW experience, and CHW baseline performance for monthly quantity of care (number of household visits). With regression analysis, we used a difference-in-difference model to estimate the effect of the intervention on monthly quantity, timeliness (percentage of children under five treated within 24 hours of symptom onset), and quality (percentage of children under five treated without protocol error) of care over a six-month post-intervention period relative to a three-month pre-intervention period. RESULTS: Use of the Dashboard during monthly supervision significantly increased the mean number of home visits by 39.94 visits per month (95% CI = 3.56-76.3; P = 0.031). Estimated effects on secondary outcomes of timeliness and quality were positive but not statistically significant. Across both study arms, CHW quantity, timeliness, and quality of care significantly improved over the study period, during which time all CHWs received dedicated monthly supervision, although effects plateaued over time. CONCLUSIONS: Our findings suggest that dedicated monthly supervision and personalised feedback using performance dashboards can increase CHW productivity. Further operational research is needed to understand how to sustain the performance improvements over time. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03684551).

Using the Lives Saved Tool to aid country planning in meeting mortality targets: a case study from Mali.

BACKGROUND: Mali is one of four countries implementing a National Evaluation Platform (NEP) to build local capacity to answer evaluation questions for maternal, newborn, child health and nutrition (MNCH&N). In 2014-15, NEP-Mali addressed questions about the potential impact of Mali's MNCH&N plans and strategies, and identified priority interventions to achieve targeted mortality reductions. METHODS: The NEP-Mali team modeled the potential impact of three intervention packages in the Lives Saved Tool (LiST) from 2014 to 2023. One projection included the interventions and targets from Mali's ten-year health strategy (PDDSS) for 2014-2023, and two others modeled intervention packages that included scale up of antenatal, intrapartum, and curative interventions, as well as reductions in stunting and wasting. We modeled the change in maternal, newborn and under-five mortality rates under these three projections, as well as the number of lives saved, overall and by intervention. RESULTS: If Mali were to achieve the MNCH&N coverage targets from its health strategy, under-5 mortality would be reduced from 121 per 1000 live births to 93 per 1000, far from the target of 69 deaths per 1000. Projections 1 and 2 produced estimated mortality reductions from 121 deaths per 1000 to 70 and 68 deaths per 1000, respectively. With respect to neonatal mortality, the mortality rate would be reduced from 39 to 32 deaths per 1000 live births under the current health strategy, and to 25 per 1000 under projections 1 and 2. CONCLUSIONS: This study revealed that achieving the coverage targets for the MNCH&N interventions in the 2014-23 PDDSS would likely not allow Mali to achieve its mortality targets. The NEP-Mali team was able to identify two packages of MNCH&N interventions (and targets) that achieved under-5 and neonatal mortality rates at, or very near, the PDDSS targets. The Malian Ministry of Health and Public Hygiene is using these results to revise its plans and strategies.

Alcohol drinking and second primary cancer risk in patients with upper aerodigestive tract cancers: a systematic review and meta-analysis of observational studies.

BACKGROUND: We conducted a systematic review and meta-analysis of existing data from observational studies to assess the strength of the association of alcohol drinking with second primary cancer risk in patients with upper aerodigestive tract (UADT; oral cavity, pharynx, larynx, and esophagus) cancer. METHODS: PubMed and Embase were searched up to July 2012 and the reference lists of studies included in the analysis were examined. Random-effects models were used to estimate summary relative risks (RR) and 95% confidence interval (CI). RESULTS: Nineteen studies, 8 cohort and 11 case-control studies, were included. In highest versus lowest meta-analyses, alcohol drinking was associated with significantly increased risk of UADT second primary cancers (RR, 2.97; 95% CI, 1.96-4.50). Significantly increased risks were also observed for UADT and lung combined (RR, 1.90; 95% CI, 1.16-3.11) and all sites (RR, 1.60; 95% CI, 1.22-2.10) second primary cancers. For an increase in the alcohol intake of 10 grams per day, dose-response meta-analysis resulted in a significantly increased RR of 1.09 (95% CI, 1.04-1.14) for UADT second primary cancers. CONCLUSIONS: Alcohol drinking in patients with UADT cancer is associated with an increased risk of second primary cancers. Studies conducted in alcohol drinking patients with UADT cancer and evaluating the effect of alcohol cessation on second primary cancer and other outcomes are needed. IMPACT: Our results emphasize the importance of prevention policies aiming to reduce alcohol drinking. Health-care professionals should encourage alcohol drinking patients with UADT cancer to reduce their consumption and reinforce the surveillance of this at-risk subpopulation.

Contribution of oenocytes and pheromones to courtship behaviour in Drosophila.

BACKGROUND: In Drosophila, cuticular sex pheromones are long-chain unsaturated hydrocarbons synthesized from fatty acid precursors in epidermal cells called oenocytes. The species D. melanogaster shows sex pheromone dimorphism, with high levels of monoenes in males, and of dienes in females. Some biosynthesis enzymes are expressed both in fat body and oenocytes, rendering it difficult to estimate the exact role of oenocytes and of the transport of fatty acids from fat body to oenocytes in pheromone elaboration. To address this question, we RNAi silenced two main genes of the biosynthesis pathway, desat1 and desatF, in the oenocytes of D. melanogaster, without modifying their fat body expression. RESULTS: Inactivation of desat1 in oenocytes resulted in a 96% and 78% decrease in unsaturated hydrocarbons in males and females, respectively. Female pheromones (dienes) showed a decrease of 90%. Inactivation of desatF, which is female-specific and responsible for diene formation, resulted in a dramatic loss of pheromones (-98%) paralleled with a two-fold increase in monoenes. Courtship parameters (especially courtship latency) from wild-type males were more affected by desat1 knocked-down females (courtship latency increased by four fold) than by desatF knocked-down ones (+65% of courtship latency).The number of transcripts in oenocytes was estimated at 0.32 and 0.49 attomole/microg for desat1 in males and females, respectively, about half of the total transcripts in a fly. There were only 0.06 attomole/microg desatF transcripts in females, all located in the oenocytes. CONCLUSION: Knock-down results for desat1 suggest that there must be very little transport of unsaturated precursors from fat body to the oenocytes, so pheromone synthesis occurs almost entirely through the action of biosynthesis enzymes within the oenocytes. Courtship experiments allow us to discuss the behavioral role of diene pheromones, which, under special conditions, could be replaced by monoenes in D. melanogaster. A possible explanation is given of how pheromones could have evolved in species such as D. simulans, which only synthesize monoenes.