From the January 18, 1977, special issue of {MMWR} Epidemiologic Notes and Reports Follow-up on Respiratory Illness -- Philadelphia

Last summer an outbreak of severe respiratory illness occurred in Pennsylvania chiefly among those who had attended a state American Legion convention in Philadelphia July 21-24, 1976 (MMWR 25 {30,33,34}). An estimated 180 cases including 29 deaths occurred (MMWR 25 {38}). An organism has now been isolated in yolk sacs of embryonated hens' eggs that appears to be the etiologic agent. For the purpose of this report the yolk sac isolate is being called a bacterium on the basis of its size and morphology.

The bacterium was first isolated from the lung tissues of 1 fatal case of Philadelphia respiratory disease and 1 fatal case of Broad Street pneumonia (see below) by inoculation of guinea pigs intraperitoneally. After a 1- to 2-day incubation period the guinea pigs developed a febrile illness that was characterized in most animals by watery eyes and prostration. Spleen suspensions of febrile guinea pigs were inoculated into yolk sacs of embryonated eggs from antibiotic-free chicken flocks. The embryos died after 4-6 days, and Gimenez-stained smears of the yolk sacs were found by microscopic examination to contain many bacilli. The bacilli were gram-negative and moderately pleomorphic. Surviving guinea pigs were shown by indirect immunofluorescence to have developed antibody to the yolk sac isolates. Because most bacteria when inoculated into the yolk sac kill the eggs in 1-2 days, an unusual rickettsia was suspected. The organism is bigger than a rickettsia, however, and the convalescent guinea pig sera failed to react in the complement fixation test with standard rickettsial antigens prepared from Coxiella burnetii, Rickettsia rickettsii, R. prowazekii, and R. typhi. Cultivation on sheep blood agar and Trypticase Soy Agar has been attempted at each yolk sac passage. Frequently, no growth has been observed, but yolk sacs infected with 1 isolate have sometimes given many minute colonies after 2-3 days' incubation. The slowness of growth has delayed bacteriological identification.

Evidence for the etiologic role of the yolk sac isolate in the epidemic has been obtained by indirect fluorescent antibody stains carried out by methods that are the same as those in regular use in the diagnosis of rickettsial diseases, except that the microdrops fixed to the slide were prepared from yolk sacs infected with isolate 1 and isolate 2 of the Philadelphia agent. The results in (Table_1) and (Table_2) were obtained with sera from 33 patients who were selected because they were Legionnaire delegates who were hospitalized, survived, and had radiologic evidence of pneumonia and fevers of at least 102 F; they thus represented the most typical survivors.

Table 1 shows some representative results. The sera with high titers gave bright staining at low dilutions which gradually decreased with increasing dilution. The brightness of staining and height of the titers are similar to that observed in other infectious diseases, for example, Rocky Mountain spotted fever. Patients 1, 2, and 3 had distinct increases in antibody titers, and 4 had only a 4-fold increase. The first specimen from patient 5 was already at a titer of 128, and there was no further increase between the eighth and twenty-ninth day of illness.

Table 2 summarizes the results with sera of the 33 Legionnaire patients tested to date; 29 gave results that suggest they were infected with the organism. Seroconversions were seen in 25 patients and antibody rises of more than 4-fold in 19. The maximum titers observed were 128 or greater in 26 out of 29 patients. The titers were usually low in the first week of illness, but they rose rapidly in the second and third weeks. The fact that 3 patients had no serologic response is not surprising since the cases were defined on a clinical and epidemiological basis. The staining of isolates 1 and 2 has been very similar with these sera and with the other sera reported below. Thus the 2 yolk-sac isolates are antigenically very similar if not identical.

Cases of Broad Street pneumonia represent disease clinically similar to Philadelphia respiratory disease that occurred in persons who did not attend the Convention, were within 1 block of Hotel A between July 1-August 18, but said they did not go into Hotel A during the epidemic period. Sera from 4 of the 38 such patients have been tested. Two have shown serologic conversions from titers of 16 or less to 512 or greater. Two had unchanging titers of 32 or less.

As controls for the fluorescent antibody tests, sera were tested from 40 patients unrelated to the outbreak whose specimens had been submitted for rickettsial diagnosis (Table_3). The rickettsial complement fixation tests had failed to demonstrate rickettsial antibody. The sera were first screened at a dilution of 1:32 and those with staining at this dilution were retested at dilutions of 1:16 through 1:512. Most of the titers observed with the yolk sac isolate were low. Two specimens had titers of 64, that is, they overlapped with the lowest titers observed in Legionnaire patients in Table 2. The staining at low dilutions with these sera was only 1+ bright; however, in all the seropositive Legionnaire patients in Table 2 and in the 2 Broad Street pneumonia patients who converted, fluorescence was 3-4+ bright in low dilutions.

In the outbreak, illness in conventioneers was associated with time spent in Hotel A. The incidence was directly related to time spent in the lobby. Sera were available from some hotel employees of 2 categories: those who worked in the lobby and those who worked in locations removed from the lobby (Table_4). Also shown in the table are the results with sera from a group of Pennsylvania Legionnaires who did not attend the convention. One positive titer in a hotel employee was seen, a cashier checker, who had a titer of 256. The titers with the other employees and the Legionnaires who did not attend the convention were within the range of the 40 non-epidemic sera reported in Table 3.

In 1966 an outbreak of acute pneumonia occurred at a large psychiatric hospital in the District of Columbia. There were 94 cases and 16 deaths. Acute and convalescent sera were available from 14 patients; they were also tested against the antigens from Isolate 1 and 2. Thirteen had distinct rises in titer of 8-fold or more, and 12 had titers of 128 or more. The brightness of staining and titers were the same as those seen with the Legionnaire patients.

The intensity of public interest in the Philadelphia epidemic makes it necessary to provide a factual account of these findings now. The etiology of the outbreak has been unknown. The present findings provide very strong evidence that the 2 epidemics were caused by the bacterium isolated in yolk sacs and that nearly all the cases had the same cause. The bacterium can be identified now by the characteristic disease it produces in guinea pigs, the characteristic death pattern in eggs, the at best dysgonic growth on the bacterial media tried, and by the fluorescent antibody staining results. Other, more complex explanations are possible. For example, the bacterium might be thought of as a secondary invader associated with a virus, but extensive virological searches have failed to reveal a virus and the serologic responses for the bacterium have been present in a very high percentage of the cases. There has not been time to identify the organism taxonomically. The source of the organism in the outbreak is not known, but the search should now be greatly facilitated.

Reported by the Leprosy and Rickettsia Br, Virology Div, Bur of Laboratories, CDC.

Follow-up Survey Data: In December 1976, selected survivors of Philadelphia respiratory disease and matched controls were interviewed concerning smoking habits, liquor and snack food preference, and knowledge of homemade liquor. The 56 patients selected for interview represented all hospitalized male survivors who had been delegates to the American Legion convention and were known to have developed an illness characterized by temperature of 102 F or higher and pneumonia proved by X-ray. The 56 controls were male delegates matched by age who had indicated on earlier survey that they had not been ill since the convention. The interviews were completed with 52 case-control pairs. Cigarette smoking habits at the time of the convention were the only significant associations with illness. The relative risk of illness among cigarette smokers was 3.4 compared to non-smokers (X2(1) = 5.5, pless than .05, McNemar) (Table_5). Cases also smoked more cigarettes and were more likely to have smoked sample cigarettes available at the convention. A previous survey showed no single cigarette brand common among cases. Pipe or cigar smoking was not associated with illness.

Reported by RG Sharrar, MD, City of Philadelphia Dept of Public Health; E Streiff, RN, MPH, Allegheny County Dept of Health; WE Parkin, DVM, Acting State Epidemiologist, Pennsylvania State Dept of Health; Bur of Epidemiology and Bur of Laboratories, CDC.

Editorial Note

Editorial Note: Editorial Note -- 1997: A bacterial etiology was not initially evident during the field investigation phase of the Legionnaires disease outbreak. Chest radiographs of case- patients revealed an interstitial pneumonia, which at that time was considered indicative of a viral infection. Because the Legionnaires disease bacterium is refractory to most stains, no bacteria could be visualized when sections of lung tissues from deceased patients were stained by commonly used methods, such as the Brown-Brenn technique. Additionally, no species of bacteria was reproducibly isolated from autopsy materials or clinical specimens because the special nutritional requirements of the Legionnaires disease bacterium precluded its growth on conventional culture media. The bacterium was, however, isolated in guinea pigs and in the yolk sacs of embry- onated hens' eggs and was visualized by Gimenez stain during one of several efforts to isolate Q fever rickettsiae (Coxiella burnetii) from specimens of lung tissue collected at autopsy. It subsequently was cultivated on enriched Mueller-Hinton agar using heavily infected yolk sacs as inoculum. The unique nutritional requirements of the bacterium were identified in separate studies, and a new culture medium was developed that now allows routine isolation of the Legionnaires disease bacterium from clinical specimens (1).

Determination of the phenotypic and genotypic properties of the Philadelphia isolate indicated that it was a novel species (Legionella pneumophila) (2). The genus Legionella now comprises approximately 40 named species and subspecies that are associated with water. Approximately half of the species have been implicated in human disease; L. pneumophila serotype 1, the prototype strain that was isolated following the Philadelphia outbreak, is responsible for most infections.

The epidemic of pneumonia that followed the American Legion convention in August 1976 was one of the most publicized epidemics in which CDC had participated. Daily newspaper reports contained "body counts," rumors of biological and chemical warfare, and accusations of cover-up by CDC. Reports by CDC in the MMWR, however, were limited to short back-page accounts, reporting that the epidemic had occurred and was under investigation.

On Friday, January 14, 1977, the director of CDC's Laboratory Division, Charles Shepard, M.D., and microbiologist Joseph E. McDade, Ph.D., went to the office of the CDC Director, David J. Sencer, M.D. After a few hesitant moments, they informed him that they had isolated the agent that had caused the outbreak. Dr. Shepard wanted to take the weekend to redo the isolation in a laboratory where they had not been working to rule out any possibility of contamination.

Although Dr. Shepard did not want to release the information until it was published in the peer-reviewed scientific literature, Dr. Sencer wanted to fulfill CDC's responsibility to immediately release the information to state and local health departments because the outbreak had been in the national news for months and this information could prevent other cases. A solution was found: MMWR is a scientific publication, and CDC published and printed MMWR. CDC could print a special edition on the following Tuesday, January 18, 1977 (normal publication was on Thursdays). Once it was in print at 1 p.m., it could be given to the news media with an embargo until 3 p.m.

Tuesday morning, as the presses were beginning to run 180,000 copies, Dr. Shepard reported that he had retrieved serum specimens from the serum bank of two earlier unsolved outbreaks of pneumonia, and they were positive for the identical organism. The presses were stopped, and the changes were made.

At 1 p.m., a conference call was scheduled from CDC to the state health officers, the Surgeon General, the National Institutes of Health, and other public health officials participating in the investigation. CDC employees who had worked in any way on Legionnaires disease -- from dishwashers in the laboratory to the chiefs of epidemiology and the laboratory -- were invited to this conference call. Following that call, CDC conducted a press conference, in which Drs. Shepard and McDade presented the findings and distributed the MMWR. This is the only occasion on which an extra issue of the MMWR (weekly) has been published.

Legionnaires disease is only one of many new diseases, syndromes, or etiologic agents that have been identified during the past 2 decades (3), and CDC has responded to these new challenges. Other noteworthy examples include Lyme disease, human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), toxic-shock syndrome, human ehrlichiosis, hantavirus pulmonary syndrome, hepatitis C virus, and Escherichia coli O157. In some instances (for example, HIV/AIDS), both the disease and its etiologic agent previously were unknown to medical science. In others, the disease already existed but was unrecognized. Legionnaires disease apparently occurred sporadically as early as the 1940s. Retrospective analysis of a "rickettsia-like agent," which was isolated from a pneumonia patient in 1947, revealed that this agent was identical to L. pneumophila (4). However, at the time of its isolation, the source of the bacterium was erroneously attributed to the guinea pigs that were used in the isolation procedure. Since the 1940s, other technologic changes, including the introduction of air-conditioning cooling towers, have facilitated the potential for exposure through dissemination of infectious aerosols of Legionella in contaminated water (5). This realization has lead to changes in routine maintenance procedures for many aerosol-producing devices such as cooling towers, spas, and respiratory therapy equipment. The development of prevention strategies is an ongoing process involving medical professionals, engineers, and chemical disinfectant manufacturers.

Identification of new etiologic agents undoubtedly will continue. For example, of the approximately 4 million cases of pneumonia that occur in the United States each year, the etiologic agent remains unidentified in up to 50% of cases even when an etiology is actively sought (6). Similarly, no etiologic agent is found in 60% of reported foodborne disease outbreaks, most of which are studied using routine diagnostic methods, nor in 32% of diarrheal outbreaks on cruise ships, despite intensive investigation (7,8). However, in contrast to the Legionnaires disease investigation, during which the etiologic agent was identified serendipitously, new molecular techniques allow for a more systematic search for infectious etiologies. In particular, the extreme sensitivity of representational difference analysis and consensus sequence-based polymerase chain reaction technology should allow the identification of many etiologic agents that previously have been refractory to culture (9). Editorial Note by: David J Sencer, MD, former Director, Center for Disease Control. Joseph E McDade, PhD, Associate Director for Laboratory Science, National Center for Infectious Diseases, CDC.

References

1. Feeley J, Gibson RJ, Gorman GW, et al. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol 1979;10:437-41.

2. Brenner DJ, Steigerwalt AG, McDade JE. Classification of the Legionnaires' disease bacterium: Legionella pneumophila, genus novum, species nova, of the family Legionellaceae, familia nova. Ann Intern Med 1979;90:656-8.

3. Institute of Medicine. Emerging infections: microbial threats to health in the United States. Washington, DC: National Academy Press, 1992.

4. McDade JE, Brenner DJ, Bozeman FM. Legionnaires' disease bacterium isolated in 1947. Ann Intern Med 1979;90:659-61.

5. Breiman RF. Impact of technology on the emergence of infectious diseases. Epidem Rev 1996;18:4-9.

6. Marston BJ. Epidemiology of community-acquired pneumonia. Infectious Diseases in Clinical Practice 1995;4(suppl 4):S232-S239.

7. Bean NH, Goulding JS, Lao C, Angulo FJ. Surveillance for foodborne-disease outbreaks -- United States, 1988-1992. In: CDC surveillance summaries (October). MMWR 1996;45 (no. SS-5).

8. Koo D, Maloney K, Tauxe R. Epidemiology of diarrheal disease outbreaks on cruise ships, 1986 through 1993. JAMA 1996;275:545-7.

9. Gao SJ, Moore PS. Molecular approaches to the identification of unculturable infectious agents. Emerging Infectious Diseases 1996;2:159-67.

TABLE 1. Results with indirect fluorescent antibody stains of the agent cultivated
in yolk sacs. Sera from selected patients with Philadelphia respiratory disease.
====================================================================================================================================================
                         Day of                                                                                                        Interpre-
Patient     Specimen     Disease       16 (a)         32            64           128            256           512          Titer        tation (b)
--------------------------------------------------------------------------------------------------------------------------------------------------
   1           S1            1          1+/1+ (c)  +/- / +/-       0/0           0/0            0/0           0/0          16/16 (d)
               S2           22          3+/3+        3+/3+        2+/3+         1+/2+        +/- /1+          0/0         128/256          C
   2           S1            4        +/- / +/-       0/0          0/0           0/0            0/0           0/0         <16/<16
               S2           11          2+/3+        2+/2+        1+/2+         1+/1+        +/- / +/-        0/0         128/128
               S3           25          3+/3+        2+/2+        2+/2+         1+/1+        +/- /1+       +/- / +/-      128/256          C
   3           S1           24        +/- / +/-    +/- / +/-       0/0           0/0            0/0           0/0         <16/<16
               S2           34          3+/3+        3+/3+        2+/3+         2+/3+          1+/2+         1+/1+       >512/>512         C
   4           S2           12          3+/3+        2+/2+        1+/1+       +/- / +/-         0/0           0/0          64/64
               S3           33          3+/3+        2+/2+        1+/2+         1+/1+        +/- /1+          0/0         128/256          C
   5           S1            8          2+/2+        2+/2+        1+/1+         1+/1+        +/- / +/-        0/0         128/128
               S3           29          3+/3+        3+/3+        2+/3+        2+ / +/-      +/- /0           0/0         128/64           P
   6           S1            5           0/0          0/0          0/0           0/0            0/0           0/0         <16/<16
               S3           29           0/0          0/0          0/0           0/0            0/0           0/0         <16/<16          N
--------------------------------------------------------------------------------------------------------------------------------------------------
(a) The reciprocal of the dilution is shown in this and other tables.
(b) C = seroconversion or increase in titer of at least 4-fold with 1 or both antigens. P = classified
    as positive because the titer was high but showed little change. N = classified as negative
    because all specimens had low titers.
(c) Brightness of staining: 0 = no staining, +/- = questionable staining, 1+ = barely detectable but
    definite staining, 2+ and 3+ = increasing brightness. The 2 numbers refer to staining of the
    bacteria in yolk sacs infected with the first 2 isolates.
(d) Highest dilution with definite staining with either antigen.
====================================================================================================================================================

TABLE 2. Results with indirect fluorescent antibody stains of the agent cultivated in
yolk sacs: Summary of results with patients with Philadelphia respiratory disease
=======================================================================================
                                                    Number of
Interpretation of Titers                            Patients
-------------------------------------------------------------
Seroconversions: >4-fold                               19
                  4-fold                                5
Positive (>=64) without seroconversion                  5
Negative                                                4

Maximum titer observed
  with seroconversions
  and positives                            8192         1
                                           2048         1
                                           1024         3
                                           >512         3
                                            512         6
                                            256         6
                                            128         6
                                             64         3

Negatives                                   <64         4
-------------------------------------------------------------
Total patients tested                                  33
=======================================================================================

TABLE 3. Results with sera from control patients
clinically suspected to have Rickettsia infections
==================================================
        Titer                   Number of Persons
--------------------------------------------------
           64                           2
           32                           6
           16                           3
          <32                          28
          <16                           1
--------------------------------------------------
        TOTAL                          40
==================================================

TABLE 4. Serologic results with other persons who did not meet clinical criteria for a
case of Philadelphia respiratory disease (a)
======================================================================================================
                                                       Titers
                                   <16     16     32     64     128     256     Total
-------------------------------------------------------------------------------------
Hotel employee, lobby                       1      2                               3
Hotel employee, non-lobby           6       4      2                     1        13
Legionnaire, not at convention      3       5      1      2                       11
-------------------------------------------------------------------------------------
(a) In all these sera the staining, even at low dilutions, was not more than 1+ (barely detectable).
======================================================================================================

TABLE 5. History of cigarette smoking at the American Legion Convention,
Philadelphia, July 1976 among case-control pairs
===========================================================================
                                     Cases
                         -------------------------------
        Controls           Smoker           Non-smoker           Total
---------------------------------------------------------------------------
          Smoker               14                    5              19
      Non-smoker               17                   16              33

           Total               31                   21              52
===========================================================================

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