Abstract:
The present invention relates to methods for determining predisposition to acute life threatening episodes (ALTE) and/or sudden infant death syndrome (SIDS) and, in particular, to methods of assessing potential susceptibility to development of ALTE and/or SIDS by determining a subject&#39;s total IgA and/or IgA1.

Description:
TECHNICAL FIELD  
       [0001]     The present invention relates to methods for determining predisposition to acute life threatening episodes (ALTE) and/or sudden infant death syndrome (SIDS) and in particular to methods of assessing potential susceptibility to development of ALTE and/or SIDS by determining a subject&#39;s total IgA and/or IgA1.  
       BACKGROUND  
       [0002]     A great deal has been done to minimize the risk of SIDS by non-specific methods related to infant care. However, prevention using specific assays related to causal mechanisms has not been explored. Identifying a causal mechanism may be expected to make a major impact on SIDS outcome through general awareness, and if used in conjunction with non-specific nursing care. The development of new techniques for identifying infants at risk of SIDS could be a significant outcome.  
         [0003]     Interest in this approach to the prevention of SIDS arose as a result of an unusual opportunity of observing a ‘prospective’ case of SIDS during a study of 250 normal infants [1]. The infants were followed from birth, measuring parameters of immune status in saliva. The key observation in the one child who died from SIDS was an extraordinarily high IgM level appearing after a mild respiratory tract infection, several weeks before the child suddenly died. While all parameters tested in the SIDS victim (i.e. albumin, IgG and IgA) were in excess of the 90th percentile level, relative levels of IgM were the highest, being more than three times the level of the 90th percentile figure (compared to approximately one and a half times the 90th percentile level for albumin, IgG and IgA). This observation in a single case was consistent with post mortem studies showing large numbers of IgM containing plasma cells in the trachea and gut of subjects dying from SIDS [2-5]. The level of IgM was much in excess of any small increases seen in matched infection control studies [6]. These observations raised the possibility that assay of IgM in saliva of infants with an upper respiratory tract infection may be a very useful marker of risk of SIDS, reflecting the disturbed mucosal immunoregulation that underpins the risk.  
         [0004]     The numerous epidemiological studies of SIDS have identified many of the risk factors of SIDS but have failed to find a cause [7]. The role of infection and disturbed immunity has been proposed as one of the potential mechanisms for SIDS [8]. The common findings at autopsy of SIDS infants are consistent with infection or inflammation as a contributing cause of death [9]. SIDS has been reported to occur after a mild upper respiratory tract infection (URTI) [9-12], however there is no evidence that favours infections by any virulent pathogen. A low grade pathogen, that results in overstimulation of the immune system may be one important link in the chain of events that culminates in respiratory arrest.  
         [0005]     There is evidence from post mortem studies [2-5, 13-14] and a prospective case study [1] of a gross disturbance of mucosal immunity in SIDS associated with prior respiratory illness or inflammation. These studies suggest an infective agent is responsible for the disturbance observed in the immune parameters which thus provides a clinical “trigger” for testing the infant for risk.  
         [0006]     Infants presenting with episodes of apnoea from which the infant recovers are termed acute life threatening episodes (ALTEs) and are classified as “near-miss” SIDS when no underlying medical condition is identified. ALTE children could, therefore, be expected to have a similar pattern of dysregulation of mucosal immunity to SIDS children.  
         [0007]     To date, however, no method exists by which a prediction of the potential susceptibility to development of ALTE and/or SIDS can be carried out on the basis of a specific immunological assay.  
         [0008]     It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.  
       SUMMARY OF THE INVENTION  
       [0009]     As indicated above, the prior art [1] pointed towards IgM levels after URTI infection as being a potentially useful parameter for study in ALTE/SIDS research. However, it has been unexpectedly found that IgA levels were significantly and consistently higher in ALTE or “near miss” SIDS cases. IgA can therefore be used as a predictor of susceptibility to development of ALTE and/or SIDS.  
         [0010]     According to a first aspect, the present invention provides a method of assessing potential susceptibility to development of ALTE and/or SIDS in a subject including:  
         [0000]     a) determination of the immunoglogulin A (IgA) level in a sample from the subject; and  
         [0000]     b) prediction of susceptibility to development of ALTE and/or SIDS by comparison of said IgA level with a predetermined standard.  
         [0011]     According to a second aspect, the present invention provides a method of assessing potential susceptibility to development of ALTE and/or SIDS in a subject including:  
         [0000]     a) determination of immunoglobulin A1 (IgA1) level in a sample from the subject; and  
         [0000]     b) prediction of susceptibility to development of ALTE and/or SIDS by comparison of said IgA1 level with a predetermined standard.  
         [0012]     Preferably, the subject is a human infant.  
         [0013]     Preferably, the sample is a sample from a subject at the time of, or any time up to approximately 2 weeks after, an upper respiratory tract infection (URTI) or symptoms.  
         [0014]     Preferably, the immunoglobulin is secretory immunoglobulin. Preferably, the secretory immunoglobulin is salivary immunoglobulin. Preferably, the sample is whole unstimulated saliva. However, it will be clear to the skilled addressee that other body secretions known to contain IgA and/or IgA1 would also be useful as samples for the present method.  
         [0015]     Preferably, the subject is not fasting when the sample is collected.  
         [0016]     Preferably, the immunoglobulin level is determined by ELISA. However, it will be understood that the immunoglobulin level may be determined by radial immunodiffusion and/or similar methods, all of which would be known to a skilled addressee. The method is particularly suitable for an assay in which the immunoglobulin level is analysed by a rapid, near-subject assay. It can thus provide a yes/no test for immediate action.  
         [0017]     In one embodiment, the immunoglobulin level is determined by contacting a body secretion with an assay device or system on a support. The sample need not necessarily be removed from the subject but the method may be applied in situ. For example, the immunoglobulin level may be analysed by contacting an assay device or system with the saliva of the subject in situ.  
         [0018]     The predetermined standard may be any appropriate standard, for example, a normal population standard or an internal personal standard. The skilled addressee will recognize the types of standards which will be useful in the present invention.  
         [0019]     In a third aspect, the present invention provides a method for assessing potential susceptibility to development of ALTE and/or SIDS in an infant including:  
         [0000]     (a) determination of the immunoglobulin A (IgA) and/or immunoglobulin A1 (IgA1) level in a sample of the infant&#39;s whole, unstimulated saliva; and  
         [0000]     (b) prediction of susceptibility to development of ALTE and/or SIDS by comparison of said IgA and/or said IgA1 level with a predetermined standard.  
         [0020]     It will be clear to the skilled addressee that the determination of the level of IgA or IgA1 could also be used as a predictor of susceptibility to development of ALTE and/or SIDS in conjunction with other indices such as other immunoglobulins, for example IgM or IgG, acute phase reactants or cellular components.  
         [0021]     In a fourth aspect, the present invention provides a kit when used in a method according to any one of the first to third aspects.  
         [0022]     In the context of the present invention, the word “standard” includes within its meaning, but is not limited to, the normal population level of immunoglobulin i.e. the average IgA or IgA1 value for age-matched normal subjects. It may also be an internal personal standard i.e. the level of IgA or IgA1 in a sample taken previously from the same individual. 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0023]      FIG. 1  IgA concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—initial sample.  
         [0024]      FIG. 2  IgM concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—initial sample.  
         [0025]      FIG. 3  IgG concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—initial sample.  
         [0026]      FIG. 4  IgA concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—14 day sample. Subject RO3 was assessed 12 days post infection.  
         [0027]      FIG. 5  IgM concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—14 day sample. Subject RO9 was assessed 14 days post immunisation with triple antigen and  Haemophilus influenzae  B.  
         [0028]      FIG. 6  IgG concentration levels (mg/L) for ALTE (▪), “mild” (◯) and well (▴) infants—14 day sample.  
         [0029]      FIG. 7A  IgA1 concentration levels (mg/L) for ALTE, “mild” and well infants.  
         [0030]      FIG. 7B  Data of  FIG. 7A  shown with 95% confidence intervals.  
         [0031]      FIG. 8  Total IgA concentration levels (mg/L) for ALTE, “mild” and well infants in which IgA1 concentration levels (mg/L) is shown in  FIGS. 7A and 7B . 
     
    
     DESCRIPTION OF THE INVENTION  
       [0032]     A preferred embodiment of the invention will now be described, by way of example only.  
       Example 1  
     Total IgA, IgG and IgM Levels in Saliva of Infants with ALTE  
       [0000]     Saliva Collection  
         [0033]     Whole mixed saliva was collected by gentle suction from the buccal cavity of the mouth [15]. This technique is successful in children (aged from 1 day) and adults [1,16].  
         [0000]     Questionnaire  
         [0034]     A standardised questionnaire was used to collect the relevant SIDS related demographics. The classification into the “near-miss” SIDS group (ALTE) was made by the attending paediatrician on the basis of clinical investigations.  
         [0000]     Saliva Tests  
         [0035]     Salivary immunoglobulins were measured by ELISA and albumin by rate nephelometry (Beckman. ARRAY) [16].  
         [0000]     Statistical Analysis  
         [0036]     The differences in mucosal immune parameters was determined between the ALTE infants and two control groups of subjects (mild URTI and well infants) using analysis of variance (ANOVA) or the appropriate non-parametric statistics.  
         [0000]     Subjects  
         [0037]     37 subjects aged 1-10 months were recruited (20 males, 17 female) in 3 categories: 
        Acute life Threatening episodes (ALTE) at John Hunter Hospital (n=5)     Mild respiratory tract illness (MILD) from General Practitioners (n=11)     A well control group (WELL) from immunisation clinics (n=21). 
 
 Questionnaire Data 
    There were more males (n=4) than females (n=1) in the ALTE group.     There were no significant differences between the groups for age, birth history, family demographics, ethnic background or family history of SIDS.     There were a higher percentage of children exposed to passive tobacco smoke (60%, n=3) in the ALTE group compared to the MILD (36%, n=4) and WELL (10%, n=2) control groups (p=0.03).     The ALTE group had a higher percentage of families in the average-below average socioeconomic category (100%) compared to the other control groups (p&lt;0.01).     There were no differences between the groups for feeding history, immunisation status, sleeping position.        
 
         [0046]     In 4 of the 5 ALTE subjects an Upper Respiratory Tract Illness (URTI) was suspected as the cause of the ALTE (Table 1).  
                                                                                                                 TABLE 1                           Doctor Questionnaire - ALTE                                Q40   Follow Up           IgA (mg/L)   Q12   Q32   Q34   URTI   Suspected            Study       14   Face   Prior   Passive   Sus-   Clinical       Number   Initial   Day   Covered   URTI   Smoke   pected   Cause                    A01   115.5   56.1   N   N   Y   N   Gastro-                                   esophageal                                   reflux       A02   228.9   22.8   N   Y   Y   Y   RSV + ve                                   Bronchiolitis       A03   410.6   230.9   N   Y   Y   Y   RSV + ve                                   Bronchiolitis       A04   91.0   28.9   Y   Y   N   Y   RSV + ve                                   Bronchiolitis       A05   26.6   79.2   N   Y   N   Y   Reflux with                                   aspiration                  
 
 Salivary Immunoglobulins 
 
         [0047]     Two samples of saliva were collected from each subject. The first sample was collected from the ALTE group within 24 hours of admission to hospital and from the MILD respiratory illness group within 48 hours of presentation of their General Practitioners. The WELL group were recruited from immunisation clinics and saliva collected at ages to approximate the ages of presentation of the ALTE and MILD groups. The second sample was collected 14 days later from each subject.  
         [0048]     The figures in Appendix C have the age related 5 th -95 th  percentile reference ranges indicated for each salivary immunoglobulin over the first year of life. 
        The salivary IgA, IgG and IgM concentration in the ALTE group were all significantly higher than the MILD (Tables 2A and 2B) and WELL (Tables 3A and 3B) groups for both sample 1 and 2 ( FIGS. 1 and 2 ).     There were no significant differences between the MILD and WELL groups for either sample 1 or sample 2 (Tables 2C and 3C).     There were two subjects in the MILD group who had grossly elevated salivary immunoglobulin concentrations in the 14 day collections. (See Appendix C). 
            RO3 had an elevated IgA 12 days post infection.    
               
 
         [0053]     RO9 had an elevated IgM that is most likely accounted for by immunisation with Triple antigen and  Haemophilus influenzae  B 14 days prior to the saliva collection.  
                                                                                                     TABLE 2A                           First Sample       Analysis of Immunoglobulins - ALTE vs MILD                ALTE   MILD                    N   Median   Range   N   Medial   Range   P-value                        IgA   5   115.55   (27-411)   11   9.93   (0-37)   &lt;0.01       IgG   5   9.21   (0-16)   11   0.00   (0-3)    0.02       IgM   5   4.61   (3-24)   11   2.18   (0-16)   0.04                  
 
         [0054]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2B 
               
             
             
               
                   
               
               
                   
               
               
                 First Sample 
               
               
                 Analysis of Immunoglobulins - ALTE vs WELL 
               
             
          
           
               
                   
                 ALTE 
                 WELL 
                   
               
             
          
           
               
                   
                 N 
                 Median 
                 Range 
                 N 
                 Medial 
                 Range 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                 IgA 
                 5 
                 115.55 
                 (27-411) 
                 21 
                 11.37 
                 (0-67) 
                 &lt;0.01 
               
               
                 IgG 
                 5 
                 9.21 
                 (0-16) 
                 21 
                 0.00 
                 (0-8)  
                 0.01 
               
               
                 IgM 
                 5 
                 4.61 
                 (3-24) 
                 21 
                 1.00 
                 (0-33) 
                 0.01 
               
               
                   
               
             
          
         
       
     
         [0055]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2C 
               
             
             
               
                   
               
               
                   
               
               
                 First Sample 
               
               
                 Analysis of Immunoglobulins - MILD vs WELL 
               
             
          
           
               
                   
                 MILD 
                 WELL 
                   
               
             
          
           
               
                   
                 N 
                 Median 
                 Range 
                 N 
                 Medial 
                 Range 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                 IgA 
                 11 
                 9.93 
                 (0-37) 
                 &lt;0.01 
                 11.37 
                 (0-67) 
                 0.68 
               
               
                 IgG 
                 11 
                 0.00 
                 (0-3)  
                 0.02 
                 0.00 
                 (0-8)  
                 0.66 
               
               
                 IgM 
                 11 
                 2.18 
                 (0-16) 
                 0.04 
                 1.00 
                 (0-33) 
                 0.36 
               
               
                   
               
             
          
         
       
     
         [0056]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3A 
               
             
             
               
                   
               
               
                   
               
               
                 Second Sample 
               
               
                 Analysis of Immunoglobulins - ALTE vs MILD 
               
             
          
           
               
                   
                 ALTE 
                 MILD 
                   
               
             
          
           
               
                   
                 N 
                 Median 
                 Range 
                 N 
                 Medial 
                 Range 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                 IgA 
                 5 
                 56.06 
                 (23-231) 
                 11 
                 8.88 
                  (1-255) 
                 0.04 
               
               
                 IgG 
                 5 
                 2.99 
                 (2-7)  
                 11 
                 0.00 
                 (0-4)  
                 0.03 
               
               
                 IgM 
                 5 
                 9.39 
                 (2-16) 
                 11 
                 2.31 
                 (0-27) 
                 0.07 
               
               
                   
               
             
          
         
       
     
         [0057]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3B 
               
             
             
               
                   
               
               
                   
               
               
                 Second Sample 
               
               
                 Analysis of Immunoglobulins - ALTE vs WELL 
               
             
          
           
               
                   
                 ALTE 
                 WELL 
                   
               
             
          
           
               
                   
                 N 
                 Median 
                 Range 
                 N 
                 Medial 
                 Range 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                 IgA 
                 5 
                 56.06 
                 (23-231) 
                 20 
                 10.53 
                 (0-58) 
                 &lt;0.01 
               
               
                 IgG 
                 5 
                 2.99 
                 (2-7)  
                 20 
                 0.00 
                 (0-6)  
                 &lt;0.01 
               
               
                 IgM 
                 5 
                 9.39 
                 (2-16) 
                 20 
                 1.66 
                 (0-14) 
                 &lt;0.01 
               
               
                   
               
             
          
         
       
     
         [0058]                                                                                                          TABLE 3C                           Second Sample       Analysis of Immunoglobulins - ALTE vs WELL                MILD   WELL                    N   Median   Range   N   Medial   Range   P-value                        IgA   11   8.88    (1-255)   20   10.53   (0-58)   0.71       IgG   11   0.00   (0-4)    20   0.00   (0-6)    0.75       IgM   11   2.31   (0-27)   20   1.66   (0-14)   0.56                    
 Conclusions 
        The grossly elevated salivary IgA concentration in 4 of 5 ALTE subjects at presentation was not observed in the MILD or WELL control groups. Salivary IgA can therefore act as a marker for ALTE (and SIDS) in subjects presenting with an otherwise mild respiratory illness. This was an unexpected result since the prior art [1] suggested IgM would be the most useful parameter in prediction of ALTE/SIDS susceptibility.     The elevated salivary IgA and IgM concentrations in 4 of 5 ALTE support the concept of an infection or inflammatory cause in ALTE (and SIDS).     RSV positive Bronchiolitis was evident in 3 of 5 ALTE subjects.          
       Example 2  
     Total IgA and IgA1 Subclass in Saliva of Infants with ALTE  
       [0000]     Study Groups  
         [0062]     Saliva samples were collected from infants on the day of admission to hospital for an unexplained acute life-threatening episode (ALTE). The infants were included in this study if all congenital or obstructive causes of apnoea had been excluded. This group of subjects have been classified as the “near-miss SIDS” infants.  
         [0063]     Saliva was collected from age matched control subjects in two categories. Normal healthy infants were recruited from the Child Immunisation Clinics and classified as WELL infants. The second group was recruited from general practitioners, who referred infants with a mild upper respiratory infection and these infants were classified as the MILD infection control group. Saliva was collected on the day of referral with the mild infection.  
         [0000]     Laboratory Analysis  
         [0064]     Saliva samples were assayed by an Enzyme Linked Immunosorbant Assay (ELISA) to detect total IgA and IgA1 subclass antibodies. The assay uses a WHO/IUIS approved monoclonal antibody for IgA1 subclass as the capture antibody in conjunction with a polyclonal antibody-enzyme labelled detection system.  
         [0000]     Results  
         [0065]     The results indicate that the concentrations of IgA1 subclass in the saliva from infants suffering an ALTE were significantly higher than the concentrations for the infants in the control groups of normal healthy infants and those suffering a mild upper respiratory infection (p=0.009) (Table 4 and  FIGS. 7A and 7B ).  
         [0066]     The concentrations of IgA1 in saliva from the normal healthy infants were not significantly different from those with mild respiratory illnesses.  
         [0067]     Five samples were assayed from each of the three study groups: ALTE babies, babies with mild infection, and well babies. The level of IgA1 was generally much higher in the samples from the ALTE babies compared to the levels in the other two groups (p=0.009). Levels in the mild infection and well baby groups were similar.  
         [0000]     Statistical Analysis  
         [0068]     The non-parametric Kruskal-Wallis test was used to compare the distributions of IgA1 values for the three groups. The probability of the three sample groups having equal IgA1 distributions is p=0.009. Due to small sample sizes, the estimated 95% confidence intervals about the group medians are equivalent to the range (i.e. min, max) of the data  
                                                                   TABLE 1                                       IgA1 (mg/L)                group   min   max   median                            ALTE   12.27   96.35   68.35           Mild infection   2.79   9.78   9.92           Well   3.02   14.85   7.29                      
 
         [0069]     Total IgA levels were also elevated in the same infants who took part in this study ( FIG. 8 ).  
         [0000]     Conclusion  
         [0070]     IgA1 concentrations are significantly elevated in infants suffering an unexplained ALTE (4 out of 5 children). Three out of the same 5 children with ALTE were found to have elevated total IgA levels. Therefore, although it is clear that both IgA and IgA1 are useful parameters in the prediction of ALTE, IgA1 levels may be the more useful parameter. Since ALTE are classified as “near-miss” SIDS (when no other medical condition is identified), it follows that both IgA and IgA1 are also useful parameters in the prediction of SIDS.  
         [0071]     Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.  
       REFERENCES  
       [0000]    
       
          1. Gleeson M, Clancy R L, Cripps A W (1993) Mucosal immune response in a case of sudden infant death syndrome.  Paed Res  Vol 33 :No  6 554-556.  
          2. Thrane P S, Rognum T O, Brandtzaeg P (1990) Increased immune response in upper respiratory and digestive tracts in SIDS.  Lancet  1:229-230.  
          3. Forsyth K D, Weeks S C, Koh L, Skinner J, Bradley J (1989) Lung immunoglobulins in the sudden infant death syndrome.  British Medical Journal  298:23-26.  
          4. Stolenberg L, Saugstad O D, Rognum T O (1992) Sudden infant death syndrome victims show local immunoglobulin M response in tracheal wall and immunoglobulin. A response in duodenal mucosa.  Paed Res  31:372-375.  
          5. Guntheroth W G (1989) Interleukin-1 as intermediary causing prolonged sleep apnea and SIDS during respiratory infections.  Med Hypotheses  28:121-123.  
          6. Gleeson M, Dobson A J, Firman D W, Cripps A W, Clancy R L, Wlodarczyk J H, Hensley M J (1991) The variability of immunoglobulins and albumin in salivary secretions of children.  Scand J. Immunol.  33: 533-541.  
          7. Wilkinson M, Jones C (1997) Australian SIDS statistics. Australia SIDS Conference. Abstract 301.  
          8. Blackwell C C, Weir D M, Busuttil A (1997) Infectious Agents and SIDS: Analysis of risk factors and preventive measures.  SIDS and Infant Mortality . (In press).  
          9. Byart R (1997) Does SIDS exist anymore? Australian SIDS Conference. Directions of SIDS Research, Presentation.  
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          11. Hoffman J H, Damus K, Hillman L, Krongrad E (1988) Risk factors for SIDS.  Ann NY Acad Sci  533:13-20.  
          12. Nelson E A, Taylor B J, Mackay S C (1989) Childcare practices and the sudden infant death syndrome.  Aust Paed J  25:202-204.  
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          15. Ostergaard P A, Blom M (1977) Whole saliva immunoglobulin levels in 60 healthy children determined by a sensitive electroimmuno technique after prior carbamylation.  Jnr Clin Chem Clin Biochem  15:393-6.  
          16. Gleeson M, McDonald W A, Cripps A W, Pyne D B, Clancy R L, Fricker P A, Wlodarczyk J H (1995) Exercise, stress, and mucosal immunity in elite swimmers.  In Advances in Mucosal Immunology . Ed J. Mestecky et al. Plenum Press New York 571-574.