Chronic bronchitis. IV--Antibody titres to bacterial antigens during acute exacerbations.SG Dalvi, AV Pathare, GG Bhave, NM Wagle
In 49 patients of chronic bronchitis with acute exacerbation, serum antibody titres were estimated against the respective pathogen isolated and cultured from sputum of individual patients. Antibody titres to Klebsiella and Staphylococcus coagulase positive organisms were found in 28 and 25 patients respectively. Paired serum samples from 21 patients showed rising antibody titres in 17 of them, whereas the antibody titres fell in the remaining 4 patients. Notably, only 2 patients demonstrated a four fold rise or fall in the antibody titres. The significance of these findings is discussed.
Keywords: Adult, Aged, Antibodies, analysis,Antigens, Bacterial, immunology,Bronchitis, blood,etiology,immunology,Coagulase, Female, Human, India, Klebsiella, immunology,Male, Middle Age, Staphylococcus, immunology,
It is generally believed that persistant inflammation, produced by either infective or non-infective agent, seems to be responsible for the acute exacerbation in patients of chronic bronchitis. Although, majority of Western literature has incriminated H. influenzae and St. pneumoniae as the predominant pathogens,,, a number of Indian studies have shown the presence of pathogenic enterobacteriaceae, namely Klebsiella and Pseudomonas along with Staphylococcus coagulase positive microorganism, as important pathogens associated with acute exacerbation in chronic bronchitis.,
There are a number of prospective studies on chronic bronchitis.,,,, However, these were inconclusive about the association of microorganisms with the pathogenesis of acute exacerbations. Not a single study, so far, has delineated any defect, immunological or otherwise that would explain why such patients are unable to rid themselves of pathogenic microorganisms. Furthermore, a couple of studies have also demonstrated an association between viral infection and acute exacerbations of chronic bronchitis.,
It thus appears that almost all studies have tried to correlate serum and secretory immunoglobulin abnormalities to the episodes of acute exacerbation in chronic bronchitis and have ignored specific antibody titres to the common microorganisms associated with acute exacerbation. Specific serum antibodies are always produced in any infection and have been used to advantage in the laboratory diagnosis of infective agents. Furthermore, it is also sometimes difficult to isolate microorganisms in sputum either because of the inadvertent use of antibiotics, or the fastidious nature of the pathogens, or due to non-availability of quantitative sputum culture. Therefore, estimation of the specific antibody titres against the common pathogen found in acute exacerbation, especially showing a four fold rise or fall in the antibody titers, can be of diagnostic value. We thus undertook this study to evaluate the specific antibody titres in serum, against the common pathogens that we found, were responsible for acute exacerbation, in our previous studies with patients of chronic bronchitis.
The clinical material consisted of 49 patients of chronic bronchitis admitted to the medical ward during acute exacerbation. Of these 46 were males and 3 females, with ages varying between 26 and 66 years. All the patients were thoroughly examined and investigated as in the previous study.
Serum from patients was tested for specific antibody titres against the respective pathogens isolated namely, Klebsiella and Staphylococcus coagulase positive, in the individual patients. A soluble extract of washed pure cultures of the common bacterial strains isolated served as the antigen. Passive haemagglutination test as described by Onkelinx et al was used to detect the antibody titres. Paired serum samples were collected before and after putting the patients on specific therapy. The mean interval between acute and convalescent serum samples of paired sera was 10. 2 days, with a range of 7 to 21 days.
Out of the 49 exacerbations studied significant bacterial colony count was seen in 28 samples (= 104/ml for pathogens, = 107/ml for commensals). In 14 samples the bacterial growth was not significant (= 104/ml for pathogens, = 107/ml for commensals). Three samples did not show any growth, while four showed contaminants. In two patients, multiple organisms were grown in significant count. In the decreasing order of frequency, the pathogens isolated were Klebsiella (11), Staphylococcus coagulase positive (8) and Pseudomonas (3). The commensals isolated were Streptococcus viridans (5), Staphylococcus coagulase negative (2) and Achromobacter (2).
[Table - 1]shows the distribution of the antibody titres against the respective pathogens isolated. [Table - 2]shows the relation ship of paired sera between the initial (acute stage) antibody titers against Klebsiella and Staphylococcus coagulase positive as compared to their subsequent (convalescent stage) antibody titers. The apparent presence of antibodies against E.coli and Proteus are not significant and reflect the well accepted cross reactivity shown by enterobacteriaceae.,
The specific antibody titres of Klebsiella and Staphylococcus coagulase positive organisms [Table - 1], suggest that they are important pathogens involved in the pathogenesis of acute exacerbation in patients with chronic bronchitis. This is in sharp contrast to the many Western studies which have implicated H. influenzae and St. pneumoniae as important pathogens.,, Inspite of having used special media like chocolate and Muller-Hinton agar, we were unsuccessful in growing H. influenzae. This implies that either inadvertent use of broad spectrum antibiotics in these chronic sufferes might have eradicated this organism from their sputum or conversely, these organisms may be having an insignificant role in the pathogenesis of acute exacerbation in our circumstances, as has been reported by other Indian workers., However, since we did not use media with X and V factors it is still possible that we may have failed to encourage its growth, given the fastidious nature of H. influenzae.
Reichek et al studying the relationship of antibody titres of exacerbation found that of the 56 exacerbations studied, 21 were associated with either a four fold rise or fall in the antibody titres. However, Gump et al in their study, found only one instance with a four fold rise in the antibody titre amongst the 22 exacerbations studied. In both these studies anti-H.influenzae antibody titres were studied. However, what is important is the timing of the estimation of the antibody titres in relation to the duration of the acute exacerbation. Unless the antibody titers are estimated early in the illness and thereafter within 2 or 3 weeks interval, a four fold rise in the antibody titres is unlikely to be seen. Interestingly, although we had shown significant growth of Klebsiella in 11 patients out of a total of 49 patients studied, a four fold rise or fall in the antibody titers were seen in only two patients. Both these were instances of anti-Klebsiella antibodies. These observations raise serious doubts regarding the role played by micro-organisms in initiating acute exacerbations of chronic bronchitis. In fact contribution of infections initiating acute exacerbations is reported to be only 45%, which further implies that non-infective agents probably play a more important role in the genesis of acute exacerbation of chronic bronchitis.
In our study, antibody titres to Klebsiella and to Staphylococcus coagulase positive organisms were found in 28 and 25 patients, whereas culture was positive in only 11 and 8 patients respectively [Table - 1]. However, in all our patients in whom we cultured a pathogenic micro-organism, an elevation in the respective antibody titer was always found. Some patients grew these pathogens in significant counts even at the end of 3 weeks of subjective and objective clinical improvement. Also, the demonstrated rise in the antibody titres in some patients receiving appropriate antibiotic therapy does not imply that the therapy was ineffective. It may be that the immune response was initiated before the patients sought medical advice. Both these observations suggest that there is no relationship between the presence of antibody in serum and the recovery rates of micro-organisms from sputum cultures.
Analysis of paired serum samples for antibody titers which were done in 21 patients revealed that 17 patients showed a rise in their antibody titres, whereas 4 patients showed falling titers [Table - 2]. A striking pattern was noticed when the relationship of initial antibody titres was studied in comparison to the paired convalescent serum antibody titres. Out of the 16 patients who had an initial titer less than 1:16, 8 patients (all culture positive) showed a subsequent rise in the anti-Klebsiella antibody titres. In contrast, in the remaining 12 patients (3 culture positive) who had an initial antibody titre above 1:16, only 2 patients each showed a rise (both culture positive) and fall (one culture positive) in their antibody titres respectively. Similarly, in the 16 patients who had an initial antibody titre against Staphylococcus coagulase positive organism of less than 1:16, 7 patients (all culture positive) showed a rise in their antibody titers. In contrast, in the remaining 9 patients no patient showed a rise in their antibody titres, whereas, 2 patients (one culture positive) showed a fall in their antibody titres.
It is thus evident that increase in the antibody titres were seen primarily in patients with low initial antibody titres, whereas patients with high initial titres predominantly showed a fall in their convalescent titers. These observations confirms that infection due to Klebsiella and Staphylococcus coagulase positive organisms does have some role in the pathogenesis of acute exacerbations in chronic bronchitis. However, it is uncertain, whether these serum antibody titres have any protective value, especially against infection which is so well localised to the bronchial tree. Nevertheless, these elevated antibody titres are definitely of diagnostic importance, however estimation of specific IgA antibody titres in the sputum, would certainly have been more useful.
A review of literature reveals that Glynn using haemagglutination test for specific antibodies against H.influenzae, found a rise in antibody titres in only one patient amongst 14 exacerbations studied. Morgan and Wood using complement fixing antibody test could not demonstrate a single rise in the antibody titres in their patients. Ross et al who also used complement fixing antibody test found no change in the antibody titers to H.influenzae but they reported an elevated initial antibody titers in 18 patients amongst the 74 exacerbations they studied. Reichek et al found that of the 56 exacerbations they studied, 21 were associated with rise in the antibody titers.
Of the 49 exacerbations studied, we found a rise or fall in the antibody titres in a total of 21 patients, of whom 12 and 9 were associated with antibody against Klebsiella and Staphylococcus coagulase positive microorganism respectively [Table - 2]. The antibody titres in our patients correlated well with the culture findings. These observations are significant, because not only did we isolate and culture distinctly different microbial flora in patients of chronic bronchitis with acute exacerbations, than that reported in the Western literature, but we also demonstrated significant rise in the antibody titres against the same pathogens. These findings merit further confirmation by a larger study taking due precautions and using highly selective media favouring the growth of H.influenzae.
Our demonstration of antibody titers against Klebsiella and Staphylococcus coagulase positive organisms in serum of patients persistently harbouring these organisms in their sputum raises the question as to why this antibody is ineffective in ridding the host of these pathogens. Gump et al have demonstrated that majority of this antibody is of the IgA type. Tomasi believes that IgA cannot mediate the complement-dependent bactericidal system. It is thought to enhance phagocytosis of pathogens by the macrophage. Medici and Buergi observed that a deficiency in the "secretory piece" of IgA exist in patients of chronic bronchitis, but Green considers cell dysfunction rather than failure of secretory antibodies as characteristic of chronic bronchitis. Furthermore, it is also known that mucocilliary clearance of particulate matter is deficient in patients with chronic bronchitis.
Thus, there are a host of factors which interplay in a complex manner. We believe that the outcome of these interactions, result in the repeated episodes of acute exacerbation in patients of chronic bronchitis. Also, rather than looking at one or two individual factors, one must consider the total effect of all these factors taken together when evaluating the pathogenesis of acute exacerbation in chronic bronchitis.
We wish to thank the Dean, Seth G. S. Medical College and K. E. M. Hospital, Bombay, for permitting us to publish this paper.
[Table - 1], [Table - 2]