key: cord-0007393-x6mbefh8 authors: Smith, Charles B.; Golden, Carole; Klauber, Melville R.; Kanner, Richard; Renzetti, Attilio title: Interactions between Viruses and Bacteria in Patients with Chronic Bronchitis date: 1976-12-03 journal: J Infect Dis DOI: 10.1093/infdis/134.6.552 sha: e0dcb2b122a77d3631b8c178cd1cc3d597b7ab3d doc_id: 7393 cord_uid: x6mbefh8 The possibility that viral infections of the respiratory tract might predispose to bacterial colonization or infection was studied in 120 patients with chronic obstructive pulmonary disease and 30 control subjects; these individuals were observed for seven years. The ratio of the number of observed to the number of expected associations between viruses and bacteria was 2.43 (P = 0.037) for the pair influenza virus and Streptococcus pneumoniae and was 2.06 (P = 0.056) for influenza virus and Haemophilus influenzae. Consistently positive, but not significant, associations were detected between rhinovirus and herpes simplex virus infections and isolations of S. pneumoniae and H. influenzae. In contrast, isolations of the nonpathogenic Haemophilus parainfluenzae could not be related to prior viral infections. Significant rises in titer of antibody to H. iniluenzae were detected on 76 occasions, and 20 (26%) of these antibody rises were associated with viral or mycoplasmal infections during the preceding 120 days. The expected number of such associations was 8.34 (ratio of number observed to number expected, 2.40; P = 0.08). These results suggest that viral infections of the respiratory tract in patients with chronic obstructive pulmonary disease are associated with increased colonization by potentially pathogenic bacteria and may also predispose to infection with H. injluenxae. The possibility that viral infections of the respiratory tract might predispose to bacterial colonization or infection was studied in 120 patients with chronic obstructive pulmonary disease and 30 control subjects; these individuals were observed for seven years. The ratio of the number of observed to the number of expected associations between viruses and bacteria was 2.43 (P = 0.037) for the pair influenza virus and Streptococcus pneumoniae and was 2.06 (P = 0.056) for influenza virus and Haemophilus influenzae. Consistently positive, but not significant, associations were detected between rhinovirus and herpes simplex virus infections and isolations of S. pneumoniae and H. influenzae. In contrast, isolations of the nonpathogenic Haemophilus parainfluenzae could not be related to prior viral infections. Significant rises in titer of antibody to H. iniluenzae were detected on 76 occasions, and 20 (26%) of these antibody rises were associated with viral or mycoplasmal infections during the preceding 120 days. The expected number of such associations was 8.34 (ratio of number observed to number expected, 2.40; P = 0.08). These results suggest that viral infections of the respiratory tract in patients with chronic obstructive pulmonary disease are associated with increased colonization by potentially pathogenic bacteria and may also predispose to infection with H. injluenxae. The concept that viral infections of the respiratory tract may impair host defenses in a manner that would lead to increased colonization or infection with pathogenic bacteria has been supported by numerous laboratory and clinical studies [1] [2] [3] [4] [5] [6] [7] . Patients with chronic obstructive pulmonary disease (COPD) characteristically suffer from recurrent acute and chronic infections due to viruses and bacteria [8, 9] , and it has been postulated that interactions between viruses and bacteria may be important in the pathogenesis of this disease [10] [11] [12] . Nevertheless, the subject of viral-bacterial interactions in this population of patients has received little investigative attention. Occasional associations between viral and bacterial infections in patients with chronic bronchitis were noted by Fisher et al. [13J and by Lambert and Stern [14] ; however, both studies were too limited in scope to allow assessment of the importance of viral-bacterial interactions in this population of patients. In 1968, we initiated a seven-year study of the role of infection in the pathogenesis of COPD. One hundred twenty patients with COPD and thirty control patients were monitored at bimonthly intervals for evidence of acute respiratory illness and infection with bacteria, viruses, and mycoplasmas. For this report we have analyzed the results of 273 viral infections and of >'1,000 bacterial cultures to assess the possibility that viral infections might predispose to bacterial colonization and/or infection in patients with COPD. One hundred twenty patients with COPD and thirty healthy control subjects were monitored eluring the seven-year period of 1968-1974 for evidence of bacterial, viral, and mycoplasmal infections. The diagnosis of COPD was made on the basis of a ratio of forced expiratory volume/sec to forced expiratory vital capacity (FEVr/FVC) of <69% and a history compatible with this symptom complex. Each patient was seen in the clinic on a bimonthly schedule; during these visits he or she was questioned regarding the occurrence of acute respiratory illness, immunizations, drug therapy, and disease status. During each clinic visit serum was obtained for antibody studies; throat swabs, nasal secretions, and sputum samples were collected for isolation of viruses and bacteria. In addition, study subjects were instructed to call the investigators at the first sign of acute respiratory illness, and this method of surveillance was amplified by a weekly telephone call to each subject. ':\Then acute respiratory illness occurred, a nurse visited the home to obtain samples of respiratory tract secretions for culture. Throat swabs were placed in Stuarts' transport medium for bacterial culture or in viral transport medium, which consisted of veal infusion broth (Difco, Detroit, Mich.) containing 1C;~bovine serum albumin, penicillin (1,000 unitsyrnl), and streptomycin (100 fLg/ml). Secretions present in the upper nasopharynx were collected for viral culture by washing of each nostril with 5 ml of 0.85% N aCI and mixing of the wash with an equal volume of virus transport medium. When available, sputum samples were also obtained for bacterial and viral culture. Sputum samples and specimens for viral culture were kept at 4 C until inoculation onto appropriate media or tissue culture cells. Inoculation of specimens was gene.rally completed within 6 hr of collection. Throat swabs and sputum samples were inoculated onto both Columbia agar containing 5% sheep's blood and peptic digest agar for isolation of bacteria. All plates were incubated in a candle jar at 37 C. Streptococcus pneumoniae was identified by demonstration of sensitivity to optochin. In the most recent 12 months, gentamicin (5 fLg/ml) was added to the Columbia agar to provide more favorable conditions for the isolation of pneumococci [15] . Haemophilus iniluenzae and Haemophilus parainfiuenzae were isolated from the peptic digest agar plates and were 553 identified by their requirement for X and/or V factors according to the method of Parker and Hoeprich [16J. Sputum, throat, and nasal wash specimens were inoculated onto human embryonic lung (WI-38) cell cultures for isolation of rhinoviruses and herpesviruses. Cell cultures were incubated at 33 C on a roller tube apparatus and were examined daily for evidence of viral CPE. Isolates from cultures exhibiting typical CPE were further characterized by tests for lability to acid and sensitivity to chloroform [17] . Tests for the presence of CF antibody to respira- Tests for CF and HAl antibodies were performed on 2,514 sam ples of sera collected from 150 patients during the seven-year period of study. A fourfold or greater rise from a previous antibody titer was considered to be evidence of infection with the test agent. Because of the known cross-reactivity between parainfluenza types I, 2, and 3, the antigen that demonstrated the highest rise in antibody titer was designated as the infecting agent. Statistical methods. The statistical analysis of viral-bacterial interactions was complicated by the fact that multiple viral and bacterial infections occurred in study patients during a short period. For this reason, there was no simple way to select a single study group (subjects with viral infection) and a control group (those free of viral infection). An additional problem in this analysis was the considerable variability in the incidence of specific viral infection (e.g., influenza) with the season of the year and from year to year. The situation was handled by an extension of the Mantel-Haenszel [22] procedure suggested by Mantel and Byar for the analysis of survival after heart transplant surgery [23] . Our adaptation of the Mantel-Byar analysis permitted us to use each study patient on multiple occasions as either a virus-positive or control (virus-negative) individual and to allow for seasonal variability in the incidence of viral infections. The prospective approach was taken, i.e., patients with, or free of, a viral infection were checked for concurrent or subsequent bacterial infection during the following seven, 30, or 60 days. Below we describe the analysis for concurrent (within seven days) infection. To control for seasonal factors, we constructed a 2 X 2 contingency negative, were selected from those who were free of virus. The requirement that control observations be made on the same day as observation of a case was too restrictive. Therefore, controls were allocated to the table containing the closest date of viral infection, but no control subject whose observation was >60 days from that of a case was included. The Mantel-Haenszel mean E (A) and variance V (A) for each table are computed in the following manner with use of the parameters defined in table 1. The ratio of the observed to expected (OlE) number of cases with both bacterial and viral infection is given by the equation OlE =~AĨ E (A) to summarize all tables. Table 2 summarizes our analysis of the association between influenza virus infection and concurrent (within seven days) isolations of S. pneumoniae. To explain the construction of table 2, we must consider the events recorded for day 395 of the study. One patient was diagnosed on that day as having influenza virus infection Since many patients received antimicrobial therapy coincident with their virus-induced exacerbations, the possibility was considered that some bacterial infections may have been suppressed, thus masking a true association between viral and bacterial infections. To compensate for this effect, in the subsequent analysis, those individuals who were receiving antibiotic therapy during or 14 days prior to the day on which a bacterial culture was taken, and whose cultures were negative for the bacteria under study, were eliminated from analysis during that time period. In the analyses described above, data from the 30 control subjects were pooled with data from the 120 patients with COPD. Separate analyses of the data from the 120 patients with COPD were performed and did not indicate an appreciable difference between the COPD group and the grou p as a whole. ed by isolation of the virus in culture (herpesvirus and rhinovirus), the date of viral culture was used. In the case of all other viral infections, demonstration of a fourfold or greater rise in titer of antibody in consecutive sera was required for diagnosis of infection. When an acute respiratory illness occurred during the same time period as the antibody rise, the date of the illness was also designated as the date of viral infection. Viral infections that were not associated with illness were not included in the analysis, leaving a total of 189 documented viral or mycoplasmal infections. To permit calculation of the rates of dual viral-bacterial infections, we included in the denominator only those viral infections that were simultaneously cultured for the presence of bacteria (table 3) . Thus, a total of 168 viral infections remained available for this analysis. A total of 102 viral infections were associated with the simultaneous isolation of S. pneumoniae, H. inlluenzae, or H. parainfluenzae from throat or sputum cultures (table 3) . The percentages of viral infections associated with simultaneous isolations of S. pneumoniae (10.7%) and H. injluenzae (13.7%) were somewhat greater than the percentages of total cultures positive for these bacteria (9.2% and 12.8%, respectively). The greatest associations of specific viral infections with isolations of these bacteria were between herpesvirus infections and isolations of S. pneumoniae (12.8%) and H. iniluenzae (21.3%) , and between influenza virus infections and isola-tions of S. pneumoniae (13.51.5 were detected on II occasions when negative cultures from patients receiving antibiotics were eliminated and on nine occasions when the cultures were kept in the analysis. Elimination of such negative cultures did have the effect of increasing the significance of some associations. For example, the greatest change occurred for the seven-day, influenza virus-S. pneumoniae analysis. The O/E ratio was 6/2.47 (P = 0.037) when negative cultures were eliminated and was 6/3.70 (P = 0.31) when all of the cultures were included. As Glasgow [25] has pointed out, differentia-tion between acquisition, colonization, invasion, and disease due to bacteria and viruses presents an important problem in clinical studies of viralbacterial interactions. The Mantel-Byar analysis permitted us to study the question of acquisition vs. chronic colonization with bacteria and indicated that the strongest association was between viral infection and bacterial colonization. On the other hand, our analysis of the relation of viral infection to subsequent rises in titer of antibody to H. inilueruae did suggest that viral infection may lead to increased invasiveness of H. injluenzae. A limitation of the Mantel-Byar method is the assumption that past history is of no significance to the current status of the patient [23] . Our consideration of prior antibiotic usage and prior colonization of the respiratory tract with the bacteria in question represents an attempt to account for those historical factors most likely to influence the results. Nevertheless, we were unable to account for other historical factors, especially previous infections, which may have occurred prior to the observation period. This situation is similar to that in any study, i.e., the results are conditional, depending on the lack of importance of any unknown factors or variables that cannot be observed. The fact that this study lasted for seven years should mitigate the effects of previous infections, and the movement of patients back and forth between study and control groups should reduce the effect of any bias based on unique past histories. With these problems of collection and interpretation of data in mind, it was possible for us to identify several instances in which viral infections were associated with an increased incidence of concurrent or subsequent colonization with bacterial pathogens. The most significant associations by our analysis were between influenza virus infections and isolations of S. pneumoniae or H. iniluerizae, Considerable data from studies in laboratory animals and populations of normal adults indicate that influenza virus infections may predispose to bacterial infection and disease. Convincing evidence linking influenza viruses and H. inilucnzae was found in Shope's studies of syn~rgy between these two agents in swine [26] and in studies with mice conducted by Sellers et a1. [3] . Pfeiffer's reports of the original isolation 559 of H. iniluenzae related this organism to epidemics of clinically significant influenza, but studies of subsequent epidemics suggested that H. iniluenzae was only an occasional secondary bacterial invader [1] . In the present study, H. injluenzae was isolated more than twice as often as expected after influenza virus infection, and the invasiveness of H. iniluenzae, as judged by rises in antibody titer, was also increased after viral infection. Despite these findings, in no instance were we able to diagnose a serious H. iniluenzae infection such as pneumonia or empyema after a viral infection, possibly because in chronic bronchitis these organisms are most often of the nonencapsulated variety and, thus, probably are inherently less pathogenic than the encapsulated .forms seen in children and normal adults [9] . Animal studies have also linked influenza virus infection with increased susceptibility to pneumococcal disease [2, 3, 34] . Finland [4] has summarized the clinical data relating these two organisms; he pointed out that in one. epidemic 50~~of patients with pneumococcal pneumonia had evidence of recent influenza virus infection. As was the case with H. iniluenzae, we were able to detect a significant association between influenza virus infection and colonization with S. pneumoniae but were not able to identify serious pneumococcal disease after influenza virus infection. Although rhinovirus infections were associated more often with isolations of S. pneumoniae and H. influenzae than would be expected, the associations were not as strong as those seen after influenza. A relation between rhinovirus infection and bacterial infections of the respiratory tract was first suggested by Cherry et a1. [6] , who reported an increased rate of isolation of S. pneumoniae and H. iniluenzae from 11 children hospitalized with rhinovirus infections. One explanation for this association is suggested by the family studies conducted by Gwaltney et a1. [15] . They noted that persons with colds were more effective transmitters of pneumococci than those without colels, and they documented simultaneous transmission of this organism and a rhinovirus by a child to other family members. A second explanation for the association between S. pneumoniae and common cold viruses is sug-gested by the studies of Webster and Clow [27] . who noted that the numbers and distribution of S. pneumoniae in the upper respiratory tract of chronic carriers increased coincident with the onset of colds. They hypothesized that respiratory viral infections may alter the milieu of the upper respiratory tract in a manner that favored increased growth of S. pneumoniae. In contrast, Foy et a1. [28] failed to detect an association between viral infections of the respiratory tract and isolations of S. pneumoniae. In a study of pneumococcal isolations from patients with pneumonia, they observed more viral infections in patients who did not harbor pneumococci (36%) than in those carrying pneumococci (26%). Possible explanations for this divergent observation include the broad age range of the patients studied and the failure to analyze the possible effects of antimicrobial therapy on the carriage of S. pneumoniae. An association between viral infections and fourfold or greater rises in titer of antibody to H. iniluenzae was detected when all of the viral and mycoplasmal infections were considered as a group. To our knowledge, a relation between viral infections of the respiratory tract and changes in titer of antibody to H. iniluenzae has not been reported; however, several authors have reported such serologic responses in patients with chronic bronchitis following acute exacerbations. most of which were probably of viral origin [29] [30] [31] . Our attempts to relate herpesvirus infection to bacterial infections suggested an association between isolation of this virus and prior (within 30 days), concurrent (seven days), and subsequent infections with both S. pneumoniae and H. in-[luenzae. In no instances, however, were the associations statistically significant. The long-held clinical impression that pneumococcal pneumonia may activate latent herpesvirus infection has recently been confirmed clinically by Fekety et a1. [7] and in the mouse by Stevens et a1. [32] . As Warren et a1. [33] pointed out several years ago. however, fever itself is an important cause of activation of latent herpesvirus infection, and the relative roles of the organism (S. pneumoniae) vs. changes in the host environment (fever) remain to be elucidated. Smith et al. Results of cultures for H. parainfluenzae were included in our analysis for purposes of comparison. Although most authors have chosen not to separate H. parainjluenzae from nonencapsulated H. influenzae [9, 13] , we elected to test each isolate for both X and V factor requirements. This separation indicated that the nonencapsulated H. iniluenzae could be related to acute illness and severity of disease in patients with COPD, whereas H. parainfluenzae was of no pathogenic significance [34] . In the present report, isolations of H. parainiluenzae were in no instance suggestively related to concurrent or prior viral infections. vVe believe that this finding adds some weight to the significance of the association of viral infections with S. pneumoniae and H. iniluenxae that were described. Our observations indicate that viral infections in patients with COPD are associated with increased rates of isolation of S. pneumoniae and H. iniluenzae, and that invasion of the latter organism, as judged by seroconversion, may also be a sequela of viral infection. 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