HK1111883B - Use of a composition for preventing or treating respiratory infections and acute otitis media in infants - Google Patents

Description

Use of a composition for preventing or treating respiratory infections and acute otitis media in infants

Background

This application has priority to U.S. provisional application 60/584,830, filed on 7/1/2004, which is incorporated herein by reference in its entirety.

(1) Field of the invention

The present invention relates generally to methods for preventing or treating respiratory infections and acute otitis media in infants.

(2) Description of the related Art

Respiratory infections are very common, especially in infants. During the first year of life, infants are prone to recurrent respiratory infections, often experiencing three to six infections in only this year. Each year in the united states alone, about 6% of infants younger than one year are hospitalized for lower respiratory tract infections.

Respiratory infections and their symptoms can range from mild to severe depending on the type of virus and the location of the infection. Upper respiratory tract infections often manifest themselves as colds, causing inflammation and swelling of the inner walls of the nose, throat and sinuses. Influenza, commonly referred to as flu, is a viral infection with high exposure of the upper respiratory tract to infection. Symptoms of influenza include fever, chills, headache, muscle aches, dizziness, cough, sore throat, runny nose, nausea and diarrhea. Another upper respiratory infection, croup, causes a very deep cough and varying degrees of dyspnea, primarily when inhaling.

It is generally accepted that lower respiratory tract infections are more severe than upper respiratory tract infections. Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract infections in infants and children younger than four years of age. VanWoensel, J., et al, Viral Lower Respiratory Tract Infection in Infants and young Children, BMJ 327: 36-40(2003). This is one such common virus that causes virtually all children three years old to become infected with RSV. In most infants and children, RSV is a mild respiratory infection that is indistinguishable from the cold. Often resulting in nasal congestion, runny nose and coughing.

Protection against RSV involves T-and B-cell responses, antibody responses (IgM, IgG, and IgA), and other immune system responses activated by bacterial and viral infections. Associations between RSV infection in infancy and the development of recurrent asthma, asthma and atopic allergy later in childhood have been proposed. Thus, limiting RSV infection can prevent serious respiratory complications that extend well into childhood.

Bronchitis is an infection of the lower respiratory tract that affects the bronchi, causing stenosis and swelling due to viral inflammation. Bronchiolitis and bronchitis are similar, but occur primarily in infants. This is an inflammation of the smaller inner diameter tubes of the bronchial branching network. This infection causes dyspnea, frequent and severe coughing and asthma, and requires hospitalization.

The perhaps most severe lower respiratory infection for infants is pneumonia. Pneumonia is caused by infections in air bubbles, causing them to become filled with fluid, usually purulent in nature, affecting the normal exchange of carbon dioxide. The severity of pneumonia depends on the amount of lung tissue involved.

Most upper and lower respiratory tract infections are caused by viruses, for which no specific prophylaxis or treatment is currently available. Some respiratory infections, including influenza, can be prevented using vaccination. However, even if vaccines for specific respiratory infections are generated, they are expensive and not universally available. Similarly, drugs to treat these infections have limited availability and are expensive. Accordingly, it would be useful to provide a non-pharmaceutical method of treating or preventing respiratory infections in infants.

Frequent respiratory infections are often associated with Acute Otitis Media (AOM), also known as middle ear infections. AOMs are characterized by acute, short-course inflammation and fluid in the middle ear. AOM is associated with rhinitis, cough, fever, sore throat, ear pain, hearing loss, irritability, allergies, absence of appetite, vomiting or diarrhea. Pus-containing otorrhea by perforating the tympanic membrane is also believed to constitute the AOM.

Fifty percent of children had at least one episode of AMO before one year of age. Eighty percent of children have at least one episode before three years of age. Between one and three years of age, 35% of children have recurrent episodes of AOM.

AOMs may be caused by viruses or bacteria. The most common bacterial strains causing AOM are Streptococcus pneumoniae (Streptococcus pneumoniae) (35% of the cases), Haemophilus influenzae (Haemophilus influenzae) (30% of the cases) and Moraxella catarrhalis (Moraxella catarrhalis) (10% of the cases). Because bacterial strains often cause infections, AOM is often treated by administration of antibiotics. In fact, more antibiotics are prescribed for AOM than for any other disease in infancy. A drawback of this broad antibiotic treatment is the development of antibiotic resistance. For example, 20% to 40% of streptococcus pneumoniae are resistant to penicillin and cefamycin. Similarly, 30% to 40% of haemophilus influenzae and 90% of moraxella catarrhalis have developed antibiotic resistance.

Due to the prevalence of antibiotic resistance in pathogenic bacteria, the American Academy of Pediatrics (American Academy of Pediatrics) and the American Academy of family physicians (American Academy of family physicians) have generated guidelines that recommend limiting the prescription of antibiotics for AOM. American Academy of Pediatrics and the American Academy of family Physician, Subcommittee on the Management of Acute Otits media, Clinical Practice guidelines (3.2004) are available in http:// www.aafp.org/PreBuilt/final _ from. pdf. Therefore, as antibiotic therapy becomes more limited, it is important to provide alternative therapies to reduce the incidence of this painful and severe condition in infants and young children.

In the analysis between the various study data (meta-analysis), the results indicated that breast-feeding had a positive effect on the frequency of respiratory infections and AOM in infants. In particular, one study showed that feeding of many currently available infant formulas was associated with a 3.6 fold increase in the hospitalization risk of respiratory infections in infants compared to exclusive breast feeding for at least four months. Bachrach, v, et al, arch, pediatr, adolesc, med.57: 237-43(2003). Furthermore, breast-fed infants have shown significantly lower AOM episodes (about 50%) than ad hoc formula-fed infants. Duffy et al, Pediatr.100 (4): e7 (1997). These differences can be attributed to the fact that human milk promotes the growth of beneficial bacteria such as Lactobacilli and Bifidobacteria. Duffy et al, dig.dis.sci 44 (8): 1499-1505(1999).

The microflora of breast-fed infants has been shown to contain predominantly bifidobacteria. In contrast, the microflora of formula fed infants is more diverse, containing bifidobacteria and Bacteroides (Bacteroides) and more pathogenic species, staphylococci (Staphylococcus), Escherichia coli (Escherichia coli) and Clostridia (clostridium). The species of bifidobacteria in breast-fed and formula-fed infants faeces are also different. Various factors have been proposed as causative agents of the different fecal flora of breast-fed and formula-fed infants, including lower levels and different compositions of proteins in human milk, lower phosphorus levels in human milk, various oligosaccharides in human milk, and hormones and cellular mediators of various immune functions in human milk. Agostoni et al, Probiotics bacteria in digital Products for Infants: nutritional by the ESPGHAN Committee on Nutrition (probiotics in infant dietary products: notes on Nutrition by the ESPGHAN Committee), j.pediatr.gastro.nutr.38: 365-. Regardless of the cause of the different bacterial populations, it is clear that breast milk has measurable benefits in the treatment or prevention of respiratory infections and AOM.

Both the american academy of pediatrics and the world health organization recommend that mothers breast feed for one to two years. However, in developed countries, these recommendations are sometimes difficult for working mothers to follow. For example, in the united states, 53% of nursing mothers introduce formula when their infants are large for one week. Before four months of age, 81% of the infants received a regular base formula. Less than 5% of us infants are breastfed at 12 months of age. Wolf.j., am.j.pub.health 93: 2000-2010(2003).

One way to promote intestinal colonization by beneficial microorganisms in formula-fed infants is by administering probiotics. Probiotics are living microorganisms that exert beneficial effects on the health of the host. Lactobacillus and bifidobacterium are among the common probiotic species. Probiotics such as these have been shown to be effective in the treatment of various gastrointestinal disorders.

For example, Reid's U.S. patent 6,613,549 relates to the use of probiotic microorganisms such as lactobacilli and bifidobacteria in the treatment of intestinal infections in infants. However, this patent does not teach any treatment of infections outside the intestinal tract. Although probiotics are effective in reducing the incidence of diarrheal disease and rotavirus shedding in hospitalized infants, probiotic combinations of bifidobacterium lactis (b.lactis) and bifidobacterium thermophilum (s.thermophilus) do not show a significant effect in terms of reducing the frequency of overall morbidity, for which health care is sought. Saavedra et al, am.j.clin.nutr.79: 261-67(2004).

U.S. patent applications 20040057965 and 20030180260 to Clancy et al describe the administration of antigens and probiotics to treat mucosal infections such as respiratory tract infections. Similarly, U.S. patent application No.20040265291 to Drake et al relates to a method of inhibiting or reducing chronic or upper respiratory infections and ear infections by administering bacteria, bacterial nutrients and antimicrobial agents. However, these references relate only to adult administration and do not disclose administration of probiotics to infants to treat respiratory infections or AOMs.

It is known that the intestinal microflora in infants is much less than that produced by adults. Although adult microflora includes more than 10¹³Individual microorganisms and up to 500 species, some are harmful and some are beneficial, and the microflora of infants is only a fraction of those microorganisms, including absolute numbers and species diversity. Since the bacterial flora and bacterial species are very different between the infant and adult intestines, it cannot be assumed that probiotic administration designed for adults is certainly beneficial for infants.

It would therefore be beneficial to provide probiotic treatment for the treatment and or prevention of respiratory infections and AOM in infants.

Summary of The Invention

Briefly, the present invention is directed to a novel method of preventing or treating respiratory infections in infants comprising administering to the infant a therapeutically effective amount of at least one bifidobacterium species in combination with at least one probiotic species that promote the growth and adherence of selected bifidobacterium strains in the small intestine. In one embodiment, the bifidobacterium species may be selected from the group of bifidobacterium species demonstrating immunomodulatory properties. These species include, for example, bifidobacterium bifidum (b.bifidum), bifidobacterium adolescentis (b.adolescentis), bifidobacterium animalis (b.animalis), bifidobacterium lactis, bifidobacterium infantis (b.infarnatis), bifidobacterium longum (b.longum) and bifidobacterium thermophilum (b.thermophilum). A particularly useful species in the present invention is Bifidobacterium lactis Bb-12.

In one embodiment, the probiotic that promotes adhesion of bifidobacteria is a member of lactobacillus, for example, lactobacillus rhamnosus GG (l.rhamnosus GG) (LGG), lactobacillus delbrueckii subsp.

The invention also relates to a novel method of preventing or treating acute otitis media in infants comprising administering to the infant a therapeutically effective amount of at least one bifidobacterium species and at least one probiotic which promotes growth and adherence of selected bifidobacterium species in the intestinal mucosa.

The present invention also relates to novel methods for preventing or treating respiratory infections and recurrent AOM infections in infants. The method comprises administering to the infant a therapeutically effective amount of at least one bifidobacterium species and at least one probiotic that promotes the growth and adherence of selected bifidobacterium species to the intestinal mucosa.

Among several advantages, it was found that obtained by the present invention is a method providing prevention or treatment of respiratory infections in infants without the need to administer unavailable or costly drugs or vaccinations. The present invention also provides methods for preventing or treating AOM without the need for administration of antibiotics that cause resistance in pathogenic bacterial species.

Brief Description of Drawings

Figure 1 is a graph showing the effect of probiotic supplementation on AOM outbreaks during the first year of life.

Fig. 2 is a graph showing the effect of probiotic supplementation on respiratory tract infections during the first year of life.

Detailed description of the preferred embodiments

Reference now will be made in detail to embodiments of the invention, one or more examples of which are set forth below. Each embodiment is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are described in or are apparent from the following detailed description. It is to be understood by one skilled in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

Definition of

As used herein, the term "treating" means slowing, ameliorating or treating a disease, disorder or disease symptom or condition.

The term "preventing" means stopping or hindering a disease, disorder or disease symptom or condition by some action.

The term "therapeutically effective amount" refers to an amount that causes amelioration or alleviation of a disease, disorder, or symptom or condition of the disease.

The term "infant" means a human less than about 2 years of age.

The term "respiratory infection" or "respiratory disease" means a disease or infection affecting the group of organs responsible for carrying oxygen in the air to the bloodstream and expelling carbon dioxide.

The term "probiotic" means a microorganism that exerts beneficial effects on the health of the host. Can be a live microbial feed supplement that beneficially affects the host by improving intestinal microbial balance, a microbial preparation containing live or dead bacteria, or a combination of both. Living organisms are generally preferred because they produce antigens of the complete system, proliferate to increase the number of such organisms in the intestinal environment to promote mucosal interactions, and can adhere to intestinal tissue to better stimulate mucosal immune responses.

The term "prebiotic" means a non-digestible food ingredient that stimulates the growth and/or activity of probiotics.

The term "recurrent" means the occurrence of 3 or more infections over the course of a year.

As used herein, the term "infant formula" means a composition that meets the nutritional needs of an infant by acting as a substitute for human milk. In the united states, the contents of infant formulas are regulated by federal regulations set forth in sections 21c.f.r.100, 106 or 107. These regulations define macronutrient, vitamin, mineral and other ingredient levels in an effort to mimic the nutritional and other characteristics of human breast milk.

Invention of the invention

According to the present invention, a new method of preventing or treating respiratory infections in infants is created. The method comprises administering to the infant a therapeutically effective amount of at least one bifidobacterium such as Bb-12 and at least one probiotic such as LGG that promotes adhesion of selected bifidobacterium strains to the intestinal mucosa.

Bifidobacteria are gram-positive anaerobic microorganisms that function in the lower half of the digestive system. They are immobile, non-sporulating and catalase negative. They have various shapes including short bent rod, bat rod and bifurcated Y-shaped rod. They are classified as lactic acid bacteria because they produce lactic acid during carbohydrate fermentation.

In one embodiment of the invention, the bifidobacteria may be selected from the group of bifidobacteria which demonstrate immunomodulatory properties. These species may include, for example, bifidobacterium bifidum, bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium lactis, bifidobacterium infantis, bifidobacterium longum and bifidobacterium thermophilum. A particularly useful bifidobacterium species in the present invention is bifidobacterium lactis Bb-12, obtained from chr.

In one embodiment of the invention, the probiotic that promotes the adhesion of bifidobacteria to the intestinal mucosa may be a member of the genus lactobacillus. Lactobacillus is a gram-positive facultative anaerobic microorganism. They do not form spores and are flagellate stalk-like or coccobacillus. Any lactobacillus species known in the art may be used in this embodiment. For example, the adhesion-promoting probiotic may be LGG, lactobacillus delbrueckii subsp bulgaricus, or a combination of the two.

LGG is a strain of lactobacillus isolated from the intestinal flora of healthy humans. Which is disclosed in U.S. patent 5,032,399 to Gorbach et al, which is incorporated herein by reference in its entirety. LGG is resistant to most antibiotics, stable in the presence of acids and bile, and adheres hot to mucosal cells of the human intestinal tract. It survives for 1 to 3 days in most individuals and up to 7 days in 30% of patients. In addition to its colonization ability, LGG beneficially affects mucosal immune responses. LGG is deposited with the American type culture for microorganisms having a deposit qualification under deposit number ATCC 53103.

Lactobacillus delbrueckii is a gram-positive, facultative anaerobic, immobile and non-sporulating, rod-shaped microorganism. Like other lactic acid bacteria, lactobacillus delbrueckii is acid-tolerant, unable to synthesize porphyrins, and has a strict fermentation mechanism with lactic acid as the major metabolic end product. The Lactobacillus delbrueckii species contains three subspecies, Lactobacillus delbrueckii subspecies delbrueckii, Lactobacillus delbrueckii subspecies L.delbrueckii subspecies and Lactobacillus delbrueckii subspecies bulgaricus.

Typically, the adhesion rate of the selected bifidobacteria to the intestinal mucosa is about 18%. Previous studies have shown that specific bacterial species can promote adhesion of bifidobacteria to the intestinal mucosa. Juntunen, m. et al, clin. diag. lab. immunol.8: 293-96(2001). In particular in the presence of two lactobacillus species, LGG (ATCC 53103) and lactobacillus delbrueckii subsp bulgaricus (obtained from Valio ltd. finland), bifidobacterial adhesion increased from 18% to 44% and 45%, respectively. As before. Without wishing to be bound by this or any other theory, it is believed that these lactobacillus strains co-aggregate and thus improve the adhesion of bifidobacteria to the intestinal mucosa and its residence time in the intestine.

According to the method of the invention, the number of respiratory tract and AOM infections in infants provided with the composition is reduced when the bifidobacterium strain is provided in combination with at least one adhesion-promoting probiotic.

In the present invention, the form of administration of the bifidobacterium strain and the adhesion promoting probiotic is not critical as long as a therapeutically effective amount is administered to the infant. More conveniently, the bifidobacterium strain and the adherence-promoting probiotic may be supplemented to infant formula which is thereafter fed to the infant.

In one embodiment, the infant formula for use in the present invention is nutritionally complete and contains suitable types and amounts of lipids, carbohydrates, proteins, vitamins and minerals. The amount of lipid or fat is generally from about 3 to about 7g/100 kal. The protein content is generally from about 1 to about 5g/100 kcal. The carbohydrate content is generally from about 8 to about 12g/100 kcal. The protein source may be any one used in the art, for example, skim milk, whey protein, casein, soy protein, hydrolyzed protein, partially hydrolyzed protein, amino acids, and the like. In one embodiment, the protein is a combination of whey protein and casein in a 60: 40 ratio. The carbohydrate source may be any of those used in the art, for example, lactose, glucose, corn syrup solids, maltodextrins, sucrose, starch, rice syrup solids, and the like. The lipid source may be any one used in the art, for example, vegetable oils such as palm oil, soybean oil, palm olein, coconut oil, medium chain triglyceride oil, high oleic sunflower oil, high oleic safflower oil, and the like.

Conveniently, commercially available infant formula is used. For example, the amino acid sequence of Enfalac,preterm formula containing ironAnd(from Mead Johnson&Company, Evansville, IN, u.s.a.) may be supplemented with appropriate amounts of bifidobacterium strains and adhesion-promoting probiotics and used IN the practice of the methods of the invention.

The infant formula of the present invention may contain ingredients designed to promote the growth of bifidobacteria in the intestinal mucosa. For example, bifidobacteria require ferrous ions, riboflavin and biotin for growth. These may be provided in combination with other ingredients in the infant formula.

As an alternative to administration of infant formula, the bifidobacterium strain and the adhesion-promoting probiotic may be administered as a supplement to the infant formula rather than entirely.

The invention may be used for the treatment or prevention of respiratory infections or AOM in infants fed exclusively breast-fed or fed a combination of breast milk and infant formula.

In a particular embodiment of the invention, at least one prebiotic may be supplemented to the infant diet in combination with a bifidobacterium strain and an adhesion promoting probiotic. In this embodiment, the prebiotic may be any prebiotic known in the art. In particular embodiments, the prebiotic is selected from the group consisting of galacto-oligosaccharide, inulin, fructo-oligosaccharide, lactulose, neosugar (neosugar), and combinations thereof.

In one embodiment of the invention, the bifidobacterium strain and the adhesion promoting probiotic are supplemented to the diet of the infant from birth until the infant is about one year old. In another embodiment of the invention the bifidobacterium strain and the adhesion promoting probiotic are supplemented into the diet of the infant from birth until the age of the infant is about three years old.

In one embodiment, the therapeutically effective amount of the bifidobacterium strain and the adhesion-promoting probiotic is about 10⁵To 10¹¹Colony forming units (cfu). In another embodiment, the therapeutically effective amount of the bifidobacterium strain and the adhesion-promoting probiotic is about 10⁶To 10⁸cfu. In one embodiment, a therapeutically effective amount is administered daily. In other embodiments, a therapeutically effective amount may be administered every other day, weekly or monthly. The frequency and size of the probiotic dose depends, for example, on the selected microorganism, the delivery vehicle and the infant to whom the medicament is administered.

It is within the level of knowledge of the skilled person to provide an increased dosage, as determined by the skilled person to be safe and effective for an individual infant. Furthermore, the minimum amounts may be varied based on their combination with prebiotic combinations and other additives that improve colonization by bifidobacteria strains.

In one embodiment of the invention, the ratio of Bb-12 to LGG is from about 10: 1 to 1: 10. In another embodiment of the present invention, the ratio of Bb-12 to LGG is from 5: 1 to 1: 5. In yet another embodiment of the present invention, the ratio of Bb-12 to LGG is from about 3: 1 to 1: 3. In a particular embodiment of the invention, the ratio of Bb-12 to LGG is about 1: 1.

The probiotic microorganisms of the present invention may be provided as a powder in capsule form, as an ingredient of an emulsion or paste, or in any other suitable carrier that is effective carrier for live microorganisms as determined by one skilled in the art. The powder composition containing the probiotic micro-organisms may be provided in a separate sachet, for example, for mixing with infant formula or primary food. For example, the capsule may be opened so that the contents may be mixed with infant formula, strained deslagged food, milk, juice, or other food for providing nutritional compositions to an infant. The emulsion or paste may also be incorporated into various food products.

In one embodiment, the bifidobacteria strain and the supplementation of adhesion-promoting probiotics prevent or treat the onset of upper respiratory infections, influenza, croup, respiratory syncytial virus, bronchitis, bronchiolitis and/or pneumonia. In another embodiment, the bifidobacterium strain and the supplementary adhesion-promoting probiotic prevent or treat the onset of AOM.

A significant reduction in the incidence of early and recurrent infections during the first year of life and antibiotic use is obtained by the specific probiotic combination of the invention. This effect is most pronounced for respiratory infections and AOM (the most common infection in infants). This is shown in figures 1 and 2. These figures illustrate that probiotic supplementation reduces the proportion of children who develop respiratory infections or AOM episodes during the first year of life. Furthermore, probiotics appear to confer protection against early infections, culminating in the fact that children develop frequent infections, including AOMs, experiencing their first infection early.

The following examples describe various embodiments of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. In the examples, all percentages are given on a weight basis unless otherwise indicated.

Example 1

This example describes the materials and methods required to demonstrate the effect of bifidobacterium strains and adhesion-promoting probiotics on respiratory tract infections and AOM frequency.

Infants participating in the double-blind placebo-controlled clinical study conducted by the inventors were recruited in a good infant clinic in tulku, finland, from 9 months to 2002 months. The only criterion included for the study was the need for infant formula two months ago. Infants with chronic disease were excluded.

In total, 81 infants were randomly grouped with individual codes and received a daily 1X 10¹⁰Colony forming units of bifidobacterium lactis Bb-12 and LGG or microcrystalline cellulose placebo until 12 months of age. Providing daily probiotic or placebo in visually identical capsules, supplementing their contents to infant formula(s) ((ii))Mead johnson nutations, Evansville, IN). The formula is used as a separate infant formula during this stage. Preventing the mother from using commercial products containing probiotics.

At 3, 7 and 12 months of age, clinical observations of the infant were made in accordance with the scheduled observations. 72 of the 81 enrolled infants completed the follow-up study (follow-up). The mean age at withdrawal of 9 infants not completed the follow-up study was 2.9 months (1.5 to 7.0), so only infants completed the study were included in the analysis.

To confirm that the probiotics remained viable during storage, microbiological analysis of random samples of the capsules was performed by microbiologists in a blind manner. It was found that for LGG and Bb-12 1X 10 for each capsule⁹To 1X 10¹⁰Survival count of (1).

All infections during the study period were recorded in a specific diary by a family or family doctor. J detail the number of respiratory tract infections, AOM diagnosed by the physician, gastrointestinal tract infections, and treatments with antibiotic agents separately. The primary measures of the study are early respiratory tract infections, physician diagnosed AOM and incidence of gastrointestinal infections. The incidence of recurrent infection (defined as 3 or more episodes) during the first year of life is considered a secondary outcome measure. It is believed that the early or recurrent need for antibiotics reflects, and is therefore documented, the suspected early or recurrent bacterial infection, respectively. Myringotomy before 12 months of age is considered to indicate frequent ear infections. Myringotomy is a surgical procedure that requires general anesthesia, but must avoid structural damage to the ear as a result of AOM.

All health issues of the study population during the first year of life were documented to distinguish between symptoms of infectious disease and non-infectious etiology. A 24-hour esophageal pH probe study was used to confirm or rule out gastro-esophageal reflux disease as a possible non-infectious cause of cough, vomiting, or increased anxiety. The diagnosis of milk allergy, a non-infectious cause of gastrointestinal or cutaneous symptoms, was confirmed by double-blind placebo-controlled milk challenge. Allergic eczema was diagnosed using the criteria introduced by Hanifin. Handin, j.m., Atopic Dermatitis in Infants and dchildern (Atopic Dermatitis in Infants and children), pediator. 763-89(1991). Specific sensitization was determined by skin puncture testing at 7 and 12 months of age. Antigens tested included banana, potato, carrot, apple. Wheat, rice, milk, egg, cod, soy and gliadin. It is believed that in the case of one or more positive reactions at any one time point, the infant is sensitized.

To determine compliance with intervention, fecal samples were again collected at registration prior to probiotic supplementation and at 3 months of age and stored at-86 ℃. Samples were obtained from 46 infants at enrollment and 45 infants at 3 months of age. Stool samples were thawed and serially diluted in phosphate buffered saline (pH 7.2, 10mM phosphate). For detection of LGG, dilutions were plated on Rogosa agar (Oxoid, Basingstoke, UK) and incubated anaerobically at 37 ℃ for four days. Typical LGG colonies were purified and DNA was extracted from the colonies. Chain identity was confirmed by polymerase chain reaction.

The data is expressed as the mean of the range or the median of the IQR to obtain an estimate of the data distribution. Test and X Using Mann-Whitney U² _{To come}A comparison of the groups between the baselines is made. Treatment groups were compared using logistic regression analysis for early infection and recurrent infection during the first 12 months of life and for recurrent need for antibiotic treatment. Analysis of recurrent infections and antibiotic treatment was performed with or without adjustment for other relevant factors. To control the relevant risk factors or mixed factors, stepwise regression analysis is performed in a forward manner. The process set is forced into a model, and other factors introduced into the model are: gender, birth mode, duration of exclusive breastfeeding, total duration of breastfeeding, elderly siblings, mother's smoking, ownership of the pet, and family history of allergies. The criteria for recording or removing variables are: the probability of F-to-recording (F-to-enter) is less than or equal to 0.10, and the probability of F-to-removing (F-to-remove) is more than or equal to 0.15. Results are given in terms of relative risk, also known as Risk Ratio (RR), and 95% Confidence Interval (CI). For times without respiratory infection and times without AOM, Kaplan-Meier curves were applied and log-rank tests were used to compare treatment groups. Data were analyzed using SPSS (version 11.5).

Example 2

This example illustrates the effect of Bb-12 and LGG on respiratory tract infection and AOM frequency. Baseline characteristics were similar in the infants receiving probiotics and placebo and are shown in table 1.

TABLE 1 Baseline characteristics and medical history

Boy	16(50％)	19(48％)
			Mean time of pregnancy (range)	39.8 weeks (36.7 to 42.1)	39.9 weeks (35.1 to 42.3)
Average birth weight (range)	3440g (2300 to 4100)	3540g (2140 to 4580)
			Senior sibling	15(47％)	24(60％)
Smoking for parents	18(56％)	22(55％)
			Special breast-feeding averages (ranges)	1.9 weeks (0.0 to 6.0)	1.9 weeks (0.0 to 6.0)
Total average (range) for breast feeding	2.0 month (0.25 to 12.0)	2.4 months (0.25 to 7.5)
			Age (range) at which intervention began	38 days (6 to65)	35 days (2 to 59)

The mean age at the start of the intervention was 38 days (range 6-65 days) in infants receiving probiotics and 35 days (range 2-59 days) in infants receiving placebo. 72 of the 81 enrolled infants (89%) completed the follow-up study. The mean age at withdrawal of 9 infants not completing the follow-up study was 2.9 months (range 1.5-7.0), so only infants completing the study were included in the analysis.

The study of probiotics reduced the risk of early respiratory infections and AOM during the first 7 months of life and the need for antibiotic treatment. These results are detailed in table 2.

TABLE 2 incidence of infection during the first 7 months of life

^＊Calibrated for maternal allergies

Calibrated for maternal allergies and birth mode

During the first 7 months of life, 25/32 (78%) infants receiving probiotics and 36/40 (90%) infants receiving placebo experienced at least one episode of acute infection. In particular, 7/32 (22%) infants receiving probiotics and 20/40 (50%) infants receiving placebo underwent AOM. 10/32 (31%) infants receiving probiotics and 24/40 (60%) infants receiving placebo were prescribed antibiotics. During the first 7 months of life, 22/32 (69%) infants receiving probiotics and 31/40 (78%) infants receiving placebo experienced at least one episode of respiratory infection. Thus, probiotic supplementation reduces the risk of early AOM, respiratory infections and antibiotic use. During the first 7 months of life, the incidence of gastrointestinal infections is low in both groups.

In addition to its effect during the first 7 months of life, probiotics reduce the incidence of recurrent infections during the first 12 months of life. These results are shown in table 3.

TABLE 3 incidence of recurrent infections and use of drug interventions during the first 12 months of Life

Calibrated for long-lived sibling and amphiphilic allergies

Calibrated for older siblings

^＊＊Calibrated for older siblings, maternal smoking, ownership of pet, duration of exclusive breastfeeding, and maternal allergies

During the first 12 months of life, 53/72 (74%) infants enrolled in the study in total experienced 3 or more infections. More specifically, 31/72 (43%) infants suffered from recurrent infection and 14/72 (19%) recurrent AOM during this phase. Furthermore, 26/72 (36%) infants experience a recurrent need for antibiotic treatment. Prebiotics significantly reduce the incidence of recurrent infections during the first 12 months of life.

For example, 9/32 (28%) infants receiving probiotics and 22/40 (55%) infants receiving placebo experienced three or more respiratory infections. Of the infants receiving probiotics, only 4/32 (13%) experienced three or more AOM episodes. In contrast, 10/40 (25%) experienced three or more AOM episodes in those infants receiving placebo. In addition, probiotic administration tends to reduce the need for myringotomy to prevent recurrent AOM or treat secretory otitis media. During the first year of life, none of the infants receiving probiotics required a myringotomy, whereas 4/40 (10%) received placebo for this procedure.

Several factors are associated with recurrent infections and the recurrent need for antibiotic therapy. Having older siblings increases the risk of recurrent respiratory infections, the recurrent need for antibiotics, and tends to increase the risk of recurrent AOM during the first 12 months of life. Family history of allergies raises the risk of recurrent infection, maternal smoking is associated with recurrent antibiotic use. The duration of the exclusive breast feeding is inversely related to the recurrent antibiotic use. Pet ownership confers protection against recurrent infections and the recurrent need for antibiotics. Thus, the effect of probiotic supplementation on the risk of recurrent infection during the first 12 months of life was calibrated for these factors.

Gastro-esophageal reflux disease was diagnosed in 1/32 (3%) infants receiving probiotics and 3/40 (8%) infants receiving placebo. None of the 32 infants receiving probiotics had cows' milk allergy compared to 3/40 (8%) infants receiving placebo. Overall, 4/32 (13%) infants receiving probiotics and 8/40 (20%) infants receiving placebo suffered from allergic eczema during the first year of life. Atopic sensitization was detected in 2/32 (6%) infants receiving probiotics and 3/40 (8%) infants receiving placebo. None of the probiotic supplemented infants in the study experienced more than 2 gastrointestinal infections during the course of the study.

The relationship between LGG fecal collection at 3 months of age and infection risk during the first 7 months of life was also evaluated. These results are presented in table 4.

TABLE 4.3 relationship between fecal recovery of LGG at month of age and infection risk during the first 7 months of life

	Probiotics (n ═ 32)	Placebo (n ═ 40)
					Positive (n ═ 23)	Negatives (n ═ 22)	RR (95％CI)
Respiratory tract infection	15(65％)	18(82％)	0.68(0.19 to 1.21) p ═ 0.31
				AOM	4(17％)	10(45％)	0.30(0.06 to 1.13) p ═ 0.057
Antibiotic use	7(30％)	12(55％)	0.46(0.12 to 1.27) p ═ 0.14
				Recurrent respiratory tract infection	1(4％)	2(9％)	0.47(0.01 to 6.95) p ═ 0.61
Recurrent AOM	0(0％)	2(9％)	0.38(N/a to 5.05) p ═ 0.23
				Recurrent antibiotic use	0(0％)	5(23％)	0.12(N/A to 0.94) p ═ 0.022

LGG in infant faeces was collected 12/46 before probiotic supplementation, 8/28 (29%) in the probiotic group, 4/18 (22%) in the placebo group. The presence of LGG in feces prior to intervention was not associated with the incidence of general infection, respiratory infection, AOM or gastrointestinal infection, nor did it have an effect on antibiotic use at any age. LGG was collected at age 3 months, i.e. after a minimum of 1 month of probiotic supplementation, in 21/28 (75%) infants receiving probiotics and 2/17 (12%) infants receiving placebo, p < 0.0001. Furthermore, the presence of LGG in the stool at this time was associated with a reduced risk of encountering at least one AMO episode before 7 months; infants were positive and negative for LGG, 4/23 (17%) and 10/22 (45%), respectively. The presence of LGG in feces at 3 months of age is also indicative of protection against recurrent infection: representation of the need for protection against LGG positivity and negativity, and antibiotic resistance relapses, in 2/23 (9%) and 10/22 (45%) infants, respectively: before 7 months, 0/23 (0%) and 5/22 (23%) infants, respectively, were positive and negative for LGG.

All references cited in this specification, including without limitation, all papers, publications, patents, patent applications, images, texts, reports, manuscripts, brochures, books, internet postings, journal articles, periodicals, and the like, are hereby incorporated by reference into this specification in their entirety. It is determined that the discussion of the references herein is merely an overview of assertions made by the authors and does not permit any reference to be made to constitute prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Further, it should be understood that features of the various embodiments may be interchanged either in whole or in part. Furthermore, those skilled in the art will recognize that the foregoing description is by way of example only, and is not intended to limit the invention more fully to that described in the appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.

Claims (7)

1. Use of a therapeutically effective amount of Bb-12 and LGG in the manufacture of a composition for preventing or treating respiratory infections in infants, wherein the therapeutically effective amount of Bb-12 and LGG is 10⁵To 10¹¹cfu, and wherein the ratio of Bb-12 to LGG is from 10: 1 to 1: 10.

2. Use according to claim 1, wherein the respiratory infection is selected from the group consisting of upper respiratory infection, influenza, croup, respiratory syncytial virus, bronchitis, bronchiolitis and pneumonia.

3. The use according to claim 1, wherein the therapeutically effective amount of Bb-12 and LGG is 10⁶To 10⁸cfu。

4. Use according to claim 1, wherein the ratio of Bb-12 to LGG is 1: 1.

5. Use of a therapeutically effective amount of Bb-12 and LGG in the manufacture of a composition for the prevention or treatment of acute otitis media in infants, wherein the therapeutically effective amount of Bb-12 and LGG is 10⁵To 10¹¹The ratio of cfu, Bb-12 to LGG is from 10: 1 to 1: 10.

6. The use according to claim 5, wherein the therapeutically effective amount of Bb-12 and LGG is 10⁶To 10⁸cfu。

7. Use according to claim 5, wherein the ratio of Bb-12 to LGG is 1: 1.