Cumulative live birth rates for women returning to ART treatment for a second ART-conceived child.

STUDY QUESTION: What are the success rates for women returning to ART treatment in the hope of having a second ART-conceived child. SUMMARY ANSWER: The cumulative live birth rate (LBR) for women returning to ART treatment was between 50.5% and 88.1% after six cycles depending on whether women commenced with a previously frozen embryo or a new ovarian stimulation cycle and the assumptions made regarding the success rates for women who dropped-out of treatment. WHAT IS KNOWN ALREADY: Previous studies have reported the cumulative LBR for the first ART-conceived child to inform patients about their chances of success. However, most couples plan to have more than one child to complete their family and, for that reason, patients commonly return to ART treatment after the birth of their first ART-conceived child. To our knowledge, there are no published data to facilitate patient counseling and clinical decision-making regarding the success rates for these patients. STUDY DESIGN, SIZE, DURATION: A population-based cohort study with 35 290 women who commenced autologous (using their own oocytes) ART treatment between January 2009 and December 2013 and achieved their first treatment-dependent live birth from treatment performed during this period. These women were then followed up for a further 2 years of treatment to December 2015, providing a minimum of 2 years and a maximum of 7 years of treatment follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cycle-specific LBR and cumulative LBR were calculated for up to six complete ART cycles (one ovarian stimulation and all associated transfers). Three cumulative LBR were calculated based on the likelihood of success in women who dropped-out of treatment (conservative, optimal and inverse probability-weighted (IPW)). A multivariable logistic regression model was used to predict the chance of returning to ART treatment for a second ART-conceived child, and a discrete time logistic regression model was used to predict the chance of achieving a second ART-conceived child up to a maximum of six complete cycles. The models were adjusted for patient characteristics and previous and current treatment characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Among the women who had their first ART-conceived live birth, 15 325 (43%) returned to treatment by December 2015. LBRs were consistently better in women who recommenced treatment with a previously frozen embryo, compared to women who underwent a new ovarian stimulation cycle. After six complete cycles, plus any surplus frozen embryos, the cumulative LBR was between 60.9% (95% CI: 60.0-61.8%) (conservative) and 88.1% (95% CI: 86.7-89.5%) (optimal) [IPW 87.2% (95% CI: 86.2-88.2%)] for women who recommenced treatment with a frozen embryo, compared to between 50.5% (95% CI: 49.0-52.0%) and 69.8% (95% CI: 67.5-72.2%) [IPW 68.1% (95% CI: 67.3-68.9%)] for those who underwent a new ovarian stimulation cycle. The adjusted odds of a second ART-conceived live birth decreased for women ≥35 years, who waited at least 3 years before returning to treatment, or who required a higher number of ovarian stimulation cycles or double embryo transfer to achieve their first child. LIMITATIONS, REASONS FOR CAUTION: Our estimates do not fully account for a number of individual prognostic factors, including duration of infertility, BMI and ovarian reserve. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to report success rates for women returning to ART treatment to have second ART-conceived child. These age-specific success rates can facilitate individualized counseling for the large number of patients hoping to have a second child using ART treatment. STUDY FUNDING/COMPETING INTEREST(S): No funding was received to undertake this study. R. Paul and O. Fitzgerald have nothing to declare. D. Lieberman reports being a fertility specialist and receiving non-financial support from MSD and Merck outside the submitted work. C. Venetis reports being a fertility specialist and receiving personal fees and non-financial support from MSD, personal fees and non-financial support from Merck Serono and Beisins and non-financial support from Ferring outside the submitted work. G.M. Chambers reports being a paid employee of the University of New South Wales, Sydney (UNSW) and Director of the National Perinatal Epidemiology and Statistics Unit (NPESU), UNSW. The Fertility Society of Australia (FSA) contracts UNSW to prepare the Australian and New Zealand Assisted Reproductive Technology Database (ANZARD) annual report series and benchmarking reports. TRIAL REGISTRATION NUMBER: NA.

Evaluating Hospital-Based Surveillance for Outbreak Detection in Bangladesh: Analysis of Healthcare Utilization Data.

BACKGROUND: The International Health Regulations outline core requirements to ensure the detection of public health threats of international concern. Assessing the capacity of surveillance systems to detect these threats is crucial for evaluating a country's ability to meet these requirements. METHODS AND FINDINGS: We propose a framework to evaluate the sensitivity and representativeness of hospital-based surveillance and apply it to severe neurological infectious diseases and fatal respiratory infectious diseases in Bangladesh. We identified cases in selected communities within surveillance hospital catchment areas using key informant and house-to-house surveys and ascertained where cases had sought care. We estimated the probability of surveillance detecting different sized outbreaks by distance from the surveillance hospital and compared characteristics of cases identified in the community and cases attending surveillance hospitals. We estimated that surveillance detected 26% (95% CI 18%-33%) of severe neurological disease cases and 18% (95% CI 16%-21%) of fatal respiratory disease cases residing at 10 km distance from a surveillance hospital. Detection probabilities decreased markedly with distance. The probability of detecting small outbreaks (three cases) dropped below 50% at distances greater than 26 km for severe neurological disease and at distances greater than 7 km for fatal respiratory disease. Characteristics of cases attending surveillance hospitals were largely representative of all cases; however, neurological disease cases aged <5 y or from the lowest socioeconomic group and fatal respiratory disease cases aged ≥60 y were underrepresented. Our estimates of outbreak detection rely on suspected cases that attend a surveillance hospital receiving laboratory confirmation of disease and being reported to the surveillance system. The extent to which this occurs will depend on disease characteristics (e.g., severity and symptom specificity) and surveillance resources. CONCLUSION: We present a new approach to evaluating the sensitivity and representativeness of hospital-based surveillance, making it possible to predict its ability to detect emerging threats.

Assisted reproductive technology in Australia and New Zealand: cumulative live birth rates as measures of success.

OBJECTIVES: To estimate cumulative live birth rates (CLBRs) following repeated assisted reproductive technology (ART) ovarian stimulation cycles, including all fresh and frozen/thaw embryo transfers (complete cycles). DESIGN, SETTING AND PARTICIPANTS: Prospective follow-up of 56 652 women commencing ART in Australian and New Zealand during 2009-2012, and followed until 2014 or the first treatment-dependent live birth. MAIN OUTCOME MEASURES: CLBRs and cycle-specific live birth rates were calculated for up to eight cycles, stratified by the age of the women (< 30, 30-34, 35-39, 40-44, > 44 years). Conservative CLBRs assumed that women discontinuing treatment had no chance of achieving a live birth if had they continued treatment; optimal CLBRs assumed that they would have had the same chance as women who continued treatment. RESULTS: The overall CLBR was 32.7% (95% CI, 32.2-33.1%) in the first cycle, rising by the eighth cycle to 54.3% (95% CI, 53.9-54.7%) (conservative) and 77.2% (95% CI, 76.5-77.9%) (optimal). The CLBR decreased with age and number of complete cycles. For women who commenced ART treatment before 30 years of age, the CLBR for the first complete cycle was 43.7% (95% CI, 42.6-44.7%), rising to 69.2% (95% CI, 68.2-70.1%) (conservative) and 92.8% (95% CI, 91.6-94.0) (optimal) for the seventh cycle. For women aged 40-44 years, the CLBR was 10.7% (95% CI, 10.1-11.3%) for the first complete cycle, rising to 21.0% (95% CI, 20.2-21.8%) (conservative) and 37.9% (95% CI, 35.9-39.9%) (optimal) for the eighth cycle. CONCLUSION: CLBRs based on complete cycles are meaningful estimates of ART success, reflecting contemporary clinical practice and encouraging safe practice. These estimates can be used when counselling patients and to inform public policy on ART treatment.

Outbreak of hepatitis E in urban Bangladesh resulting in maternal and perinatal mortality.

BACKGROUND: Hepatitis E virus (HEV) causes outbreaks of jaundice associated with maternal mortality. Four deaths among pregnant women with jaundice occurred in an urban community near Dhaka, Bangladesh, in late 2008 and were reported to authorities in January 2009. We investigated the etiology and risk factors for jaundice and death. METHODS: Field workers identified suspected cases, defined as acute onset of yellow eyes or skin, through house-to-house visits. A subset of persons with suspected HEV was tested for immunoglobulin M (IgM) antibodies to HEV to confirm infection. We used logistic regression analysis to identify risk factors for HEV disease and for death. We estimated the increased risk of perinatal mortality associated with jaundice during pregnancy. RESULTS: We identified 4751 suspected HEV cases during August 2008-January 2009, including 17 deaths. IgM antibodies to HEV were identified in 56 of 73 (77%) case-patients tested who were neighbors of the case-patients who died. HEV disease was significantly associated with drinking municipally supplied water. Death among persons with HEV disease was significantly associated with being female and taking paracetamol (acetaminophen). Among women who were pregnant, miscarriage and perinatal mortality was 2.7 times higher (95% confidence interval, 1.2-6.1) in pregnancies complicated by jaundice. CONCLUSIONS: This outbreak of HEV was likely caused by sewage contamination of the municipal water system. Longer-term efforts to improve access to safe water and license HEV vaccines are needed. However, securing resources and support for intervention will rely on convincing data about the endemic burden of HEV disease, particularly its role in maternal and perinatal mortality.

Informing an investment case for Japanese encephalitis vaccine introduction in Bangladesh.

Japanese encephalitis virus (JEV) is a major threat to human health. Bangladesh is considering introducing a JEV vaccine; however, the investment case is hampered by a limited understanding of key aspects of JEV ecology. We conducted a seroprevalence study in a high-incidence region using an assay that limits cross-reactivity with dengue virus. We also trapped mosquitoes and collected information about potential host species. We used mathematical models to recover risk factors for infection and underlying probabilities of severe disease and death. We observed 19.0% [95% confidence interval (CI):17.1 to 21.1] of JEV antibodies. On average, 0.7% (95% CI: 0.2 to 2.0) of the susceptible population gets infected yearly, with pig proximity being the main human infection risk factor. Our traps captured 10 different mosquito species that have been linked with JEV transmission. We estimated that 1 in 1000 infections results in severe disease, 1 in 10,000 results in death, and 76% of severe cases are missed by surveillance.

Hepatitis E as a cause of adult hospitalization in Bangladesh: Results from an acute jaundice surveillance study in six tertiary hospitals, 2014-2017.

In the absence of reliable data on the burden of hepatitis E virus (HEV) in high endemic countries, we established a hospital-based acute jaundice surveillance program in six tertiary hospitals in Bangladesh to estimate the burden of HEV infection among hospitalized acute jaundice patients aged ≥14 years, identify seasonal and geographic patterns in the prevalence of hepatitis E, and examine factors associated with death. We collected blood specimens from enrolled acute jaundice patients, defined as new onset of either yellow eyes or skin during the past three months of hospital admission, and tested for immunoglobulin M (IgM) antibodies against HEV, HBV and HAV. The enrolled patients were followed up three months after hospital discharge to assess their survival status; pregnant women were followed up three months after their delivery to assess pregnancy outcomes. From December'2014 to September'2017, 1925 patients with acute jaundice were enrolled; 661 (34%) had acute hepatitis E, 48 (8%) had hepatitis A, and 293 (15%) had acute hepatitis B infection. Case fatality among hepatitis E patients was 5% (28/589). Most of the hepatitis E cases were males (74%; 486/661), but case fatality was higher among females-12% (8/68) among pregnant and 8% (7/91) among non-pregnant women. Half of the patients who died with acute hepatitis E had co-infection with HAV or HBV. Of the 62 HEV infected mothers who were alive until the delivery, 9 (15%) had miscarriage/stillbirth, and of those children who were born alive, 19% (10/53) died, all within one week of birth. This study confirms that hepatitis E is the leading cause of acute jaundice, leads to hospitalizations in all regions in Bangladesh, occurs throughout the year, and is associated with considerable morbidity and mortality. Effective control measures should be taken to reduce the risk of HEV infections including improvements in water quality, sanitation and hygiene practices and the introduction of HEV vaccine to high-risk groups.

Determinants of health care utilisation: the case of Timor-Leste.

Background: Health financing and delivery reforms designed to achieve universal health coverage (UHC) need to be informed by an understanding of factors that both promote access to health care and undermine it. This study examines the level of health care utilisation in Timor-Leste and the factors that drive it. Methods: Data from a nationally representative cross-sectional survey of health care utilisation in 1712 households were used to develop multilevel models exploring how need and predisposing and enabling factors explain health care utilisation at both primary and secondary care facilities. Results: Need was found to be the key driver in seeking both primary care and hospital services. Rural households were less likely to go to hospital (odds ratio 0.7) than urban households. The poorest quintile was also less likely to use more expensive hospital services than other socio-economic groups. Conclusions: Understanding the determinants of seeking health care in Timor-Leste is of considerable policy significance, because health care is free at the point of use. Our findings indicate that the public resources for health care are subsidising the rich more than the poor. Health care reforms in Timor-Leste need to reduce the 'other' costs of health care, such as distance barriers, to address these inequities.

Incidence of Acute Diarrhea-Associated Death among Children < 5 Years of Age in Bangladesh, 2010-12.

Although acute diarrheal deaths have declined globally among children < 5 years, it may still contribute to childhood mortality as an underlying or contributing cause. The aim of this project was to estimate the incidence of acute diarrhea-associated deaths, regardless of primary cause, among children < 5 years in Bangladesh during 2010-12. We conducted a survey in 20 unions (administrative units) within the catchment areas of 10 tertiary hospitals in Bangladesh. Through social networks, our field team identified households where children < 5 years were reported to have died during 2010-12. Trained data collectors interviewed caregivers of the deceased children and recorded illness symptoms, health care seeking, and other information using an abbreviated international verbal autopsy questionnaire. We classified the deceased based upon the presence of diarrhea before death. We identified 880 deaths, of which 36 (4%) died after the development of acute diarrhea, 17 (2%) had diarrhea-only in the illness preceding death, and 19 (53%) had cough or difficulty breathing in addition to diarrhea. The estimated annual incidence of all-cause mortality in the unions < 13.6 km of the tertiary hospitals was 26 (95% confidence interval [CI] 16-37) per 1,000 live births compared with the mortality rate of 37 (95% CI 26-49) per 1,000 live births in the unions located ≥ 13.6 km. Diarrhea contributes to childhood death at a higher proportion than when considering it only as the sole underlying cause of death. These data support the use of interventions aimed at preventing acute diarrhea, especially available vaccinations for common etiologies, such as rotavirus.

A Low-Cost, Community Knowledge Approach to Estimate Maternal and Jaundice-Associated Mortality in Rural Bangladesh.

In the absence of a civil registration system, a house-to-house survey is often used to estimate cause-specific mortality in low- and middle-income countries. However, house-to-house surveys are resource and time intensive. We applied a low-cost community knowledge approach to identify maternal deaths from any cause and jaundice-associated deaths among persons aged ≥ 14 years, and stillbirths and neonatal deaths in mothers with jaundice during pregnancy in five rural communities in Bangladesh. We estimated the method's sensitivity and cost savings compared with a house-to-house survey. In the five communities with a total of 125,570 population, we identified 13 maternal deaths, 60 deaths among persons aged ≥ 14 years associated with jaundice, five neonatal deaths, and four stillbirths born to a mother with jaundice during pregnancy over the 3-year period before the survey using the community knowledge approach. The sensitivity of community knowledge method in identifying target deaths ranged from 80% for neonatal deaths to 100% for stillbirths and maternal deaths. The community knowledge approach required 36% of the staff time to undertake compared with the house-to-house survey. The community knowledge approach was less expensive but highly sensitive in identifying maternal and jaundice-associated mortality, as well as all-cause adult mortality in rural settings in Bangladesh. This method can be applied in rural settings of other low- and middle-income countries and, in conjunction with hospital-based hepatitis diagnoses, used to monitor the impact of programs to reduce the burden of cause-specific hepatitis mortality, a current World Health Organization priority.

Hepatitis B Surface Antigen Seroprevalence among Prevaccine and Vaccine Era Children in Bangladesh.

Bangladesh introduced hepatitis B vaccine in a phased manner during 2003-2005 into the routine childhood vaccination schedule. This study was designed to evaluate the impact of the introduction of hepatitis B vaccine in Bangladesh by comparing hepatitis B surface antigen (HBsAg) prevalence among children born before and after vaccine introduction and to estimate the risk of vertical transmission of chronic hepatitis B virus (HBV) infection from mother to infant. We also evaluated the field sensitivity and specificity of an HBsAg point-of-care test strip. We selected a nationally representative sample of 2,100 prevaccine era and 2,100 vaccine era children. We collected a 5-mL blood sample from each child. One drop of blood was used to perform rapid HBsAg testing. If a child had a positive HBsAg test result with the rapid test, a blood sample was collected from the mother of the HBsAg-positive child and from the mothers of two subsequently enrolled HBsAg-negative children. All samples were tested for serologic markers of HBV infection using standard enzyme-linked immunosorbent assay. One (0.05%) child in the vaccine era group and 27 (1.2%; 95% confidence interval [CI]: 0.8-1.7%) children in the prevaccine era group were HBsAg positive. Mothers of HBsAg-positive children were more likely to be HBsAg positive than mothers of HBsAg-negative children (odds ratios = 4.7; 95% CI: 1.0-21.7%). Sensitivity of the HBsAg rapid test was 91.2% (95% CI: 76.6-98.1%) and specificity was 100% (95% CI: 99.9-100%). The study results suggest that even without a birth dose, the hepatitis B vaccine program in Bangladesh was highly effective in preventing chronic HBV infection among children.

A large-scale behavior change intervention to prevent Nipah transmission in Bangladesh: components and costs.

BACKGROUND: Nipah virus infection (NiV) is a bat-borne zoonosis transmitted to humans through consumption of NiV-contaminated raw date palm sap in Bangladesh. The objective of this analysis was to measure the cost of an NiV prevention intervention and estimate the cost of scaling it up to districts where spillover had been identified. METHODS: We implemented a behavior change communication intervention in two districts, testing different approaches to reduce the risk of NiV transmission using community mobilization, interpersonal communication, posters and TV public service announcements on local television during the 2012-2014 sap harvesting seasons. In one district, we implemented a "no raw sap" approach recommending to stop drinking raw date palm sap. In another district, we implemented an "only safe sap" approach, recommending to stop drinking raw date palm sap but offering the option of drinking safe sap. This is sap covered with a barrier, locally called bana, to interrupt bats' access during collection. We conducted surveys among randomly selected respondents two months after the intervention to measure the proportion of people reached. We used an activity-based costing method to calculate the cost of the intervention. RESULTS: The implementation cost of the "no raw sap" intervention was $30,000 and the "only safe sap" intervention was $55,000. The highest cost was conducting meetings and interpersonal communication efforts. The lowest cost was broadcasting the public service announcements on local TV channels. To scale up a similar intervention in 30 districts where NiV spillover has occurred, would cost between $2.6 and $3.5 million for one season. Placing the posters would cost $96,000 and only broadcasting the public service announcement through local channels in 30 districts would cost $26,000. CONCLUSIONS: Broadcasting a TV public service announcement is a potential low cost option to advance NiV prevention. It could be supplemented with posters and targeted interpersonal communication, in districts with a high risk of NiV spillover.

A Controlled Trial to Reduce the Risk of Human Nipah Virus Exposure in Bangladesh.

Human Nipah virus (NiV) infection, often fatal in Bangladesh, is primarily transmitted by drinking raw date palm sap contaminated by Pteropus bats. We assessed the impact of a behavior change communication intervention on reducing consumption of potentially NiV-contaminated raw sap. During the 2012-2014 sap harvesting seasons, we implemented interventions in two areas and compared results with a control area. In one area, we disseminated a "do not drink raw sap" message and, in the other area, encouraged only drinking sap if it had been protected from bat contamination by a barrier ("only safe sap"). Post-intervention, 40% more respondents in both intervention areas reported knowing about a disease contracted through raw sap consumption compared with control. Reported raw sap consumption decreased in all areas. The reductions in the intervention areas were not significantly greater compared to the control. Respondents directly exposed to the "only safe sap" message were more likely to report consuming raw sap from a protected source than those with no exposure (25 vs. 15%, OR 2.0, 95% CI 1.5-2.6, P < 0.001). While the intervention increased knowledge in both intervention areas, the "only safe sap" intervention reduced exposure to potentially NiV-contaminated sap and should be considered for future dissemination.

Respiratory Viruses Associated Hospitalization among Children Aged <5 Years in Bangladesh: 2010-2014.

BACKGROUND: We combined hospital-based surveillance and health utilization survey data to estimate the incidence of respiratory viral infections associated hospitalization among children aged < 5 years in Bangladesh. METHODS: Surveillance physicians collected respiratory specimens from children aged <5 years hospitalized with respiratory illness and residing in the primary hospital catchment areas. We tested respiratory specimens for respiratory syncytial virus, parainfluenza viruses, human metapneumovirus, influenza, adenovirus and rhinoviruses using rRT-PCR. During 2013, we conducted a health utilization survey in the primary catchment areas of the hospitals to determine the proportion of all hospitalizations for respiratory illness among children aged <5 years at the surveillance hospitals during the preceding 12 months. We estimated the respiratory virus-specific incidence of hospitalization by dividing the estimated number of hospitalized children with a laboratory confirmed infection with a respiratory virus by the population aged <5 years of the catchment areas and adjusted for the proportion of children who were hospitalized at the surveillance hospitals. RESULTS: We estimated that the annual incidence per 1000 children (95% CI) of all cause associated respiratory hospitalization was 11.5 (10-12). The incidences per 1000 children (95% CI) per year for respiratory syncytial virus, parainfluenza, adenovirus, human metapneumovirus and influenza infections were 3(2-3), 0.5(0.4-0.8), 0.4 (0.3-0.6), 0.4 (0.3-0.6), and 0.4 (0.3-0.6) respectively. The incidences per 1000 children (95%CI) of rhinovirus-associated infections among hospitalized children were 5 (3-7), 2 (1-3), 1 (0.6-2), and 3 (2-4) in 2010, 2011, 2012 and 2013, respectively. CONCLUSION: Our data suggest that respiratory viruses are associated with a substantial burden of hospitalization in children aged <5 years in Bangladesh.

Raw Sap Consumption Habits and Its Association with Knowledge of Nipah Virus in Two Endemic Districts in Bangladesh.

Human Nipah virus (NiV) infection in Bangladesh is a fatal disease that can be transmitted from bats to humans who drink contaminated raw date palm sap collected overnight during the cold season. Our study aimed to understand date palm sap consumption habits of rural residents and factors associated with consumption. In November-December 2012 the field team interviewed adult respondents from randomly selected villages from Rajbari and Kushtia Districts in Bangladesh. We calculated the proportion of people who consumed raw sap and had heard about a disease from raw sap consumption. We assessed the factors associated with raw sap consumption by calculating prevalence ratios (PR) adjusted for village level clustering effects. Among the 1,777 respondents interviewed, half (50%) reported drinking raw sap during the previous sap collection season and 37% consumed raw sap at least once per month. Few respondents (5%) heard about NiV. Thirty-seven percent of respondents reported hearing about a disease transmitted through raw sap consumption, inclusive of a 10% who related it with milder illness like diarrhea, vomiting or indigestion rather than NiV. Respondents who harvested date palm trees in their household were more likely to drink sap than those who did not own date palm trees (79% vs. 65% PR 1.2, 95% CI 1.1-1.3, p<0.001). When sap was available, respondents who heard about a disease from raw sap consumption were just as likely to drink it as those who did not hear about a disease (69% vs. 67%, PR 1.0, 95% CI 0.9-1.1, p = 0.512). Respondents' knowledge of NiV was low. They might not have properly understood the risk of NiV, and were likely to drink sap when it was available. Implementing strategies to increase awareness about the risks of NiV and protect sap from bats might reduce the risk of NiV transmission.

A novel low-cost approach to estimate the incidence of Japanese encephalitis in the catchment area of three hospitals in Bangladesh.

Acute meningoencephalitis syndrome surveillance was initiated in three medical college hospitals in Bangladesh in October 2007 to identify Japanese encephalitis (JE) cases. We estimated the population-based incidence of JE in the three hospitals' catchment areas by adjusting the hospital-based crude incidence of JE by the proportion of catchment area meningoencephalitis cases who were admitted to surveillance hospitals. Instead of a traditional house-to-house survey, which is expensive for a disease with low frequency, we attempted a novel approach to identify meningoencephalitis cases in the hospital catchment area through social networks among the community residents. The estimated JE incidence was 2.7/100,000 population in Rajshahi (95% confidence interval [CI] = 1.8-4.9), 1.4 in Khulna (95% CI = 0.9-4.1), and 0.6 in Chittagong (95% CI = 0.4-0.9). Bangladesh should consider a pilot project to introduce JE vaccine in high-incidence areas.