Transfer Learning for Automated OCTA Detection of Diabetic Retinopathy.

Purpose: To test the feasibility of using deep learning for optical coherence tomography angiography (OCTA) detection of diabetic retinopathy. Methods: A deep-learning convolutional neural network (CNN) architecture, VGG16, was employed for this study. A transfer learning process was implemented to retrain the CNN for robust OCTA classification. One dataset, consisting of images of 32 healthy eyes, 75 eyes with diabetic retinopathy (DR), and 24 eyes with diabetes but no DR (NoDR), was used for training and cross-validation. A second dataset consisting of 20 NoDR and 26 DR eyes was used for external validation. To demonstrate the feasibility of using artificial intelligence (AI) screening of DR in clinical environments, the CNN was incorporated into a graphical user interface (GUI) platform. Results: With the last nine layers retrained, the CNN architecture achieved the best performance for automated OCTA classification. The cross-validation accuracy of the retrained classifier for differentiating among healthy, NoDR, and DR eyes was 87.27%, with 83.76% sensitivity and 90.82% specificity. The AUC metrics for binary classification of healthy, NoDR, and DR eyes were 0.97, 0.98, and 0.97, respectively. The GUI platform enabled easy validation of the method for AI screening of DR in a clinical environment. Conclusions: With a transfer learning process for retraining, a CNN can be used for robust OCTA classification of healthy, NoDR, and DR eyes. The AI-based OCTA classification platform may provide a practical solution to reducing the burden of experienced ophthalmologists with regard to mass screening of DR patients. Translational Relevance: Deep-learning-based OCTA classification can alleviate the need for manual graders and improve DR screening efficiency.

Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma.

BACKGROUND: The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects. METHODS: Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients. RESULTS: Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K(trans) values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1alpha, IL-8, ICAM-1, and VCAM-1. CONCLUSION: Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.

The Gambian National Impregnated Bednet Programme: costs, consequences and net cost-effectiveness.

Clinical trials have indicated that treating mosquito nets with insecticide could be a potentially cost-effective method of preventing malaria. As malaria is one of the most common causes of death in children under five in developing countries, there has been substantial interest in whether such findings can be replicated for a country's control programme in practice. The cost-effectiveness of the Gambian National Insecticide-impregnated Bednet Programme (NIBP), from the viewpoint of providers (government and non-governmental agencies) and the community, has been calculated. Information was collected from existing records, interviews with NIBP personnel, observation and household surveys. Information is provided on the resource use consequences of the NIBP in terms of reduced expenditure on anti-malaria preventive measures, treatment in government health services, household financed treatment and "charity" (burial, funeral and mourning activities), as well as cash income lost as a result of child death. The annual implementation cost of the NIBP was D757,875 (US$91,864), of which 86% was recurrent cost. The estimated number of death averted was 40.56. The net implementation cost-effectiveness ratio per death averted and discounted life years gained were D3884 (US$471) and D260 (US$31.5), respectively. Adding the cost of all mosquito nets would increase the cost-effectiveness ratios by over five times, which is an important consideration for countries with a lower coverage of mosquito nets per capita. It is concluded that insecticide-impregnated mosquito nets are one of the more efficient ways of reducing deaths in children under 10 years in rural Gambia.

The Gambian National Impregnated Bednet Programme: evaluation of the 1994 cost recovery trial.

Following the success of a controlled trial of insecticide-impregnated bednets in reducing mortality in children. The Gambia started a National Impregnated Bednet Programme (NIBP) in 1992. The objectives of this programme were to introduce impregnated bednets into all primary health care (PHC) villages and to establish a system of cost recovery over a three-year period. During the initial phase of the programme, when insecticide was given out free, a high uptake was achieved. However, after small user charges were introduced in 1993, coverage dropped to a low level. In 1994, different systems of insecticide distribution and permethrin formulations were tried in an attempt to improve coverage. A nationwide cross-sectional survey carried out during the 1994 rainy season measured coverage by distribution channel, as well as the knowledge, attitudes and practices of health workers and villagers during the intervention. Overall, only 16% of bednets were impregnated in 1994, compared to 80% when the insecticide was offered free of charge in previous years. Lack of money was the major reason given by villagers for not impregnating their bednets in 1994. Use of impregnated bednets was higher in areas where the sale of permethrin emulsion by village health workers was supplemented by the sale of insecticide in individual packages through shops. In villages where insecticide was distributed free to women with small children through governmental mother and child health (MCH) services, higher levels of coverage were achieved among women and young children than in villages where other distribution systems were used. We conclude that the sale of insecticide through the private sector may increase bednet impregnation rates in African communities, and that the free distribution of insecticide through MCH services may be an effective way of targeting young children, the group most at risk of malaria.

The Gambian National Impregnated Bed Net Programme: evaluation of effectiveness by means of case-control studies.

Two case-control studies, one on mortality and the other on malaria morbidity, were carried out in order to evaluate the impact of the Gambian National Insecticide Bed Net Programme during the second year of intervention and to explore the feasibility of such a study for the evaluation of programme effectiveness. For the mortality study, children 1-9 years old who died during the 1993 rainy season were matched by age and sex with 2 healthy controls from the same village. For the morbidity study, children 1-9 years old attending Fatoto or Jahalia Health Centres in The Gambia and who had fever and parasitaemia > or = 5000/microL were matched by age with a child attending the health centres without fever or parasitaemia. An additional healthy control was recruited from the case's village. No impact of insecticide-treated bed nets on mortality was detected and this was in keeping with the results obtained by prospective surveillance. A protective effect of insecticide-treated nets on malaria morbidity was detected when cases were compared with controls recruited at the health centres. However, this disappeared when cases were compared with controls recruited from the cases' villages. The mortality case-control study suggested that reducing the time between onset of disease and treatment may have an important impact on childhood mortality. In order to calculate programme cost-effectiveness, important for informed resource allocations to be made by health managers, it is essential to obtain evidence of effectiveness. This can be done by means of case-control studies, which are easier to carry out and require fewer resources than prospective surveillance. Nevertheless, it is necessary to be conscious of their pitfalls, particularly of the bias involved in the choice of cases and controls. The measurement of insecticide on the nets of the cases or controls is essential for such studies.

PIP: The impact of the Gambian National Impregnated Bed Net Program was evaluated during its second year of implementation through two case-control studies on malaria mortality and morbidity. The first study matched 167 children 1-9 years of age who died during the 1993 rainy season with two healthy controls (n = 334) of the same age and sex from the same village. The second study matched 143 children 1-9 years of age attending Fatoto or Jahalia Health Centers with fever or parasitemia with a child the same age attending the health centers without these symptoms and an additional healthy control from the same village. The mortality study failed to document any protective effect of insecticide-treated bed nets. Child survival was more closely linked to consultation with a village health worker during serious illness, household availability of chloroquine tablets, sponging, and maternal awareness of fever and convulsions as symptoms of malaria. A protective effect of the bed nets on malaria morbidity was revealed when cases were compared with health center controls, but this effect disappeared when cases were compared with controls from the same village. Going to bed early and father having a job other than working on the family farm were the two variables associated with significant protection against malaria in the multivariate analysis. Case-control studies are easier to conduct and require fewer resources than large prospective community-based studies. However, it is recommended that any future such studies of malaria control programs clarify the criteria used to select cases and controls and incorporate insecticide measurements of study nets.

The impact of a national impregnated bed net programme on the outcome of pregnancy in primigravidae in The Gambia.

In 1992, the Gambian national impregnated bed net programme (NIBP) introduced insecticide treatment of bed nets into half of the primary health care villages in The Gambia. One component of the evaluation of this programme was the determination of whether it had any impact on the outcome of pregnancy in primigravidae. From February 1992, 651 primigravidae were recruited into the study. Less than 50% of them used an insecticide-treated bednet. During the rainy season the prevalence of Plasmodium falciparum among primigravidae was lower, fewer babies were classified as premature, and the mean birth weight was higher in villages where treated bed nets were used than in control villages. Therefore, during the rainy season, despite the low use of insecticide-treated bed nets by Gambian primigravidae, the NIBP had some impact on the outcome of pregnancy, particularly on the percentage of premature babies, and this was probably due to the decreased risk of malaria infection achieved during this period.

Financing mechanisms for village activities in The Gambia and their implications for financing insecticide for bednet impregnation.

The recent enthusiasm for impregnated bednets as a malaria control measure leaves unresolved the question of how to finance them. The National Impregnated Bednet Programme in The Gambia faced the question of how to obtain funds from villages to finance the cost of insecticide, but knew very little about current village fundraising for development purposes. A survey was conducted of such fundraising, and questions also asked about willingness to pay for insecticide and preferred means of paying. All 53 villages surveyed paid taxes/rates, but 34% of villages reported no voluntary fundraising. The most common reason for collecting money was for the maintenance of wells (40% of villages). Collective farming was used as a means of raising money in 32% of villages. There was some variation in the type and extent of fundraising by region and also by the predominant ethnic groups of the village. Villages with voluntary fundraising activities seemed to have well established collective mechanisms for agreeing on sums to be collected and their use, and for collecting and recording income and expenditure. Non-payment was rare, and misuse of funds was not reported. Respondents were asked how much compounds might be willing to pay for insecticide impregnantion: the most frequently cited maximum amounts were D5 and 10, and minimum D1 and 5 (D15 = 1 pound). The paper discusses payment options for insecticide, such as whether the village should be allowed to decide itself how to raise funds, and whether the payment should be made only by households with nets or by a village-wide mechanism such as collective farming.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: Surveys were conducted in 53 villages in The Gambia to determine the status of current village fundraising for development purposes. Questions were also asked about respondents' willingness to pay for insecticide and the preferred means of paying. All of the surveyed villages paid taxes/rates, but 34% of villages reported no voluntary fundraising. The most common reason for collecting money was for the maintenance of wells; such fundraising was cited by 40% of villages. Collective farming was used as a means of raising money in 32% of villages. The authors identified some variation in the type and extent of fundraising by region and also by the predominant ethnic groups of the village. Villages with voluntary fundraising activities seemed to have well established collective mechanisms for agreeing upon sums to be collected and their use, and for collecting and recording income and expenditure. Non-payment was rare and the misuse of funds was not reported. Respondents most frequently cited a compound willingness to pay a maximum of D5-10 for insecticide impregnation, with D15 equivalent to one pound Sterling. Payment options for insecticide are discussed, followed by the presentation of details of the actual mechanism selected for implementation by the National Impregnated Bednet Program.

Comparison of mortality between villages with and without Primary Health Care workers in Upper River Division, The Gambia.

Health services utilization was analysed in a rural area of the The Gambia. In general, health workers were consulted frequently. However, verbal autopsies showed that children who died had rarely consulted health workers, particularly if they came from villages where such workers were not posted. Traditional healers were consulted frequently, independently of the presence of a village health worker. The relationship between cause specific mortality and the utilization of health services is discussed. Childhood mortality was similar in villages with or without a primary health care worker at the time of the study.

PIP: The study aim was to evaluate the impact of mortality and primary health care (PHC) services utilization prior to death, based on data from the mortality surveillance system in the Upper River Division (URD) of The Gambia. The sample of villages with greater than 400 persons included 355 villages with a voluntary village health workers (VHW) and/or a trained traditional birth attendant (TBA), and 9 villages without. Recording of births and deaths was accomplished by one registrar for every 200 children or under, and totaled 373 village registrars and 8 field workers covering a population of 133,000. Cause of death for children aged 5 years was determined by 3 physicians and collected for senior field assistants. Morbidity data for children was gathered from monthly forms completed by VHWs and BAs. VHWs and TBAs were trained 6=8 weeks at Basse Hospital and 4-6 weeks at Bansang Regional Hospital, respectively, with periodic retraining. Local PHC centers provided health education, environmental health education, immunization, nutrition, treatment and referral. There were 6 health centers in URD, which was the base of operations for travel to 5-6 sites 1-2 times per month for running clinics, evaluation of referred patients, and supervision of PHC activities. There were 16, 216 episodes of malaria, 6111 episodes of respiratory infection (ALRI), and 6380 episodes of acute gastroenteritis reported through the PHC system. That means .63, .23, and .25 episodes per person per year, respectively. More than 50% of cases of ALRI involved consultation with VHWs. There were 915 deaths among children aged 5 years in one year. Of the 94% reports on the deaths made by relatives, there were 85% dying at home, and 8% dying at a health center or hospitals. 13% (117) were inpatients during a portion of the precipitating illness. Survivors of illnesses were higher among those children receiving consultation with the VHW. Only 33% of children who died had consulted a VHW during the final illness. TBAs reported 50% of deaths recorded by the surveillance system. TBAs are selectively consulted.

A malaria control trial using insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of The Gambia, west Africa. 2. Mortality and morbidity from malaria in the study area.

Background data on child mortality and morbidity from malaria were obtained in a new study area in the centre of The Gambia, south of the river, chosen as the site for a malaria intervention trial. Infant and child mortality rates were 120 and 41 per 1000 respectively. Results obtained using post-mortem questionnaires suggested that malaria was an uncommon cause of death in children under the age of one year but responsible for about 40% of deaths in children aged 1-4 years. Ninety-two percent of deaths attributed to malaria occurred during or immediately after the rainy season. Parasite and spleen rates in children aged 1-5 years at the end of the malaria transmission season were 66% and 64% respectively. Malariometric indices were similar in primary health care (PHC) villages, selected as sites for an intervention with insecticide-treated bed nets and targeted chemoprophylaxis, and in smaller, non-PHC, control villages.

A malaria control trial using insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of The Gambia, west Africa. 5. Design and implementation of the trial.

A large-scale malaria intervention programme using insecticide-treated bed nets and chemoprophylaxis administered to children was introduced into a rural area of The Gambia. The operation was carried out using the existing primary health care (PHC) service in the region. Training of the village health workers, sensitization of the communities, and implementation of net impregnation and the drug delivery programme are described. This delivery system resulted in over 90% of nets being treated with insecticide and 80% of children receiving over 90% of their tablets during the rainy season. There was considerable variation in the distribution of permethrin on a bed net and between individual nets, which is likely to facilitate the spread of insecticide resistance in the local mosquito populations. Bed nets made from heavier fabrics tended to absorb more insecticide than those made from lighter materials. Four months after dipping, 89% of the insecticide had been lost from treated nets. This was probably due mainly to women washing their nets, an activity carried out on average once every 2 months during the rainy season. The high number of insecticide-treated bed nets in the study area demonstrated that a malaria control programme operated through a PHC system can be implemented successfully.

A malaria control trial using insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of The Gambia, west Africa. 6. The impact of the interventions on mortality and morbidity from malaria.

The effects of insecticide-impregnated bed nets on mortality and morbidity from malaria have been investigated during one malaria transmission season in a group of rural Gambian children aged 6 months to 5 years. Sleeping under impregnated nets was associated with an overall reduction in mortality of about 60% in children aged 1-4 years. Mortality was not reduced further by chemoprophylaxis with Maloprim given weekly by village health workers throughout the rainy season. Episodes of fever associated with malaria parasitaemia were reduced by 45% among children who slept under impregnated nets. The addition of chemoprophylaxis provided substantial additional benefit against clinical attacks of malaria; 158 episodes were recorded among 946 children who slept under impregnated nets but who also received chemoprophylaxis. Chemoprophylaxis reduced the prevalence of splenomegaly and parasitaemia at the end of the malaria transmission season by 63% and 83% respectively. Thus, insecticide-impregnated bed nets provided significant protection in children against overall mortality, mortality attributed to malaria, clinical attacks of malaria, and malaria infection. The addition of chemoprophylaxis provided substantial additional protection against clinical attacks of malaria and malaria infection but not against death.

Decline in meningococcal antibody levels in African children 5 years after vaccination and the lack of an effect of booster immunization.

Antibodies to group A meningococcal polysaccharide were measured by hemagglutination (HA) and by ELISA in sera obtained from Gambian children before vaccination and 3 weeks, 2 years, and 5 years after vaccination with a group A + group C meningococcal capsular polysaccharide vaccine. Children were 1-4 years old at the time of vaccination. Most showed a good initial response to vaccination, including those aged 1-2 years. However, antibody titers declined progressively during follow-up, and 5 years after vaccination, antibody titers measured by both HA and ELISA had returned to prevaccination levels. This decline was not influenced significantly by a booster dose of vaccine given 2 years after initial immunization. Administration of malaria chemoprophylaxis reduced the rate at which antibody levels fell after initial immunization. Sustained protection of children against group A meningococcal disease will require the development of vaccines that are immunogenic in infants and that can induce T cell memory.

Urban beri-beri in The Gambia, west Africa.

Thirty-eight patients with beri-beri (13 dry, 14 wet and 11 mixed type) were seen at the Royal Victoria Hospital, Banjul, The Gambia, in 1990-1991, with a peak in September-October (25 cases) during the latter half of the rainy season. They were all non-confined residents of urban areas. Overt risk factors were pregnancy (6), alcohol (5), fevers (4), chronic disability (4), and exercise (2). Four patients (10.5%) died. The potential for large outbreaks of beri-beri exist in many urban areas of West Africa where polished rice is the staple diet.

Risk factors for HIV-2 infection in The Gambia.

A serosurvey was carried out to assess the prevalence of HIV infection in The Gambia and the importance of possible risk factors. The overall prevalence of HIV-2 infection among the 4228 adults studied was 1.7% while that of HIV-1 was 0.1%. The prevalence of HIV-2 was similar in both sexes but higher among those greater than 25 years of age and those who lived in two small towns along a main transport route. Among men, multivariate analysis showed prevalence of HIV-2 infection was significantly greater among those in the more affluent occupations, those without a secondary education and those with a history of urethral discharge. Among women, infection was more frequent in divorcees and widows and those who had been married several times. The prevalence was also higher in individuals born in Guinea-Bissau and in an ethnic group which originated there.

PIP: Researchers took blood samples from 4228 adults aged 15 years in The Gambia to determine the prevalence of HIV-2 infection and risk factors. HIV-2 infection was more prevalent than HIV-1 infection. HIV-2 prevalence stood at 39% for females and 31% for males, but the difference was insignificant. Individuals 25 years old were more likely to be HIV-2 seropositive than those 25 (p.01). Further, HIV-2 prevalence was significantly higher in the small towns of Soma and Farafenni on the Trans-Gambia Highway than other areas of the country (p.01; 3.2% vs. 1.3% for Greater Banjul and 1.4% for the remainder of the country). It also was greater for people who had their blood samples taken at a health center than those who gave theirs elsewhere (p.01). HIV-2 infection was more prevalent for people born in Guinea- Bissau and in the Manjago tribe which originated from Guinea-Bissau than those born in The Gambia or elsewhere (p.025 for place of birth and p.01 for tribe). Marital status played an insignificant role in seropositivity for men, but divorced and widowed women had a significantly greater infection rate than other women (p.001; odds ratio [OR] 10.4 vs. 1-20). Further, infection significantly increased as the number of husbands women had had increased (p.05; OR 6.8). HIV- 2 positivity was significantly higher among women who reported using a condom at least once during the past year with casual partners (p.01; OR 16.7). Skilled manual laborers, businessmen, and traders were more likely to be infected with HIV-2 than farmers, unskilled laborers, and while collar men (p.05). Men with at least a secondary education were at significantly lower risk than men with less than a secondary education (p.01; OR .1 VS. .7-1.6). Men who had had at least 2 cases of urethral discharge had a significantly higher infection rate than those who did not (p.005; OR 4.8 vs. .8-1).

Outbreak of beri-beri in The Gambia.

In 1988, an outbreak of beri-beri occurred in The Gambia during the rainy season. At least 140 people were affected, and 22 died. A clear response to thiamine was observed in 8 patients who were in hospital, and the outbreak resolved after distribution of thiamine in the community. Gradual replacement of traditional cereals with imported milled rice may have increased susceptibility to beri-beri.

PIP: This study examined an outbreak of beri-beri which occurred in The Gambia between July and September 1988. 140 people were affected and 22 died. The outbreak resolved after distribution of thiamine in the community. Gradual replacement of traditional cereals with imported milled rice may have increased susceptibility to beri-beri. The 1st reports of beri-beri came from the village of Chilla where 25 of 975 inhabitants described their symptoms. Active case-finding located 38 affected people. All patients had peripheral edema and 1/3 had a mixed motor and sensory neuropathy. The earliest symptoms started 3 weeks after the start of the rainy season. All inhabitants were Muslim whose staple diet is millet and imported rice from Thailand. Attack rates in Chilla and in other clinics were highest in young men. Within a few days of hospitalization (8) all patients responded rapidly to treatment of oral thiamine of 10 mg/day. Since 22 people with symptoms suggestive of beri-beri had already died in the outbreak area before thiamine became available the authors chose not to assign patients to treatment and non-treatment groups. As a result of the response to treatment of the hospital patients, thiamine was distributed to health centers and village health workers in the affected areas. Daily thiamine (200 mg) was given until symptoms were resolved. The trend towards consumption of imported rice and away from other cereals has gradually reduced people's thiamine intake and thiamine stores. In the affected area, this factor, combined with the high rainfall, high agricultural workload, cooking methods, and possible thiamine antagonists, led to the outbreak.

Yellow fever in the Gambia, 1978--1979: epidemiologic aspects with observations on the occurrence of orungo virus infections.

An epidemic of yellow fever (YF) occurred in the Gambia between May 1978 and January 1979. Retrospective case-finding methods and active surveillance led to the identification of 271 clinically suspected cases. A confirmatory or presumptive laboratory diagnosis was established in 94 cases. The earliest serologically documented case occurred in June 1978, at the extreme east of the Gambia. Small numbers of cases occurred in August and September. The epidemic peaked in October, and cases continued to occur at a diminishing rate through January, when a mass vaccination campaign was completed. The outbreak was largely confined to the eastern half of the country (MacCarthy Island and Upper River Divisions). In nine survey villages in this area (total population 1,531) the attack rate was 2.6--4.4%, with a mortality rate of 0.8%, and a case fatality rate of 19.4%. If these villages are representative of the total affected region, there may have been as many as 8,400 cases and 1,600 deaths during the outbreak. The disease incidence was highest in the 0- to 9-year age group (6.7%) and decreased with advancing age to 1.7% in persons over 40 years. Overall, 32.6% of survey village inhabitants had YF complement-fixing (CF) antibodies. The prevalence of antibody patterns indicating primary YF infection decreased with age, in concert with disease incidence. The overall inapparent:apparent infection ratio was 12:1. In persons with serological responses indicating flaviviral superinfection, the inapparent:apparent infection ratio was 10 times higher than in persons with primary YF infection. Sylvatic vectors of YF virus, principally Aedes furcifer-taylori and Ae. luteocephalus are believed to have been responsible for transmission, at least at the beginning of the outbreak. Eighty-four percent of wild monkeys shot in January 1979 had YF neutralizing antibodies, and 32% had CF antibodies. Domestic Aedes aegypti were absent or present at very low indices in many severely affected villages (see companion paper). In January, however, aegypti-borne YF 2.5 months into the dry season was documented by isolation of YF virus from a sick man and from this vector species in the absence of sylvatic vectors. Thus, in villages where the classical urban vector was abundant, interhuman transmission by Ae. aegypti occurred and continued into the dry season. A mass vaccination campaign, begun in December, was completed on 25 January, with over 95% coverage of the Gambian population. A seroconversion rate of 93% was determined in a group of vaccinees. This outbreak emphasizes the continuing public health importance of YF in West Africa and points out the need for inclusion of 17D YF vaccination in future programs of multiple immunication.