Chlamydia pneumoniae, Strain TWAR

•

rev1ew Chlamydia pneumoniae, Strain TWAR*

J Thomas Grayston,

M.D.

his article will summarize information about ChlaT mydia pneumoniae, strain TWAR under the fol-

lowing headings: (I) microbiology and classification, (2) laboratory diagnosis, (3) clinical diseases, (4) antibiotic sensitivity and treatment and (5) epidemiology. A complete up-to-date bibliography is included. 1·26 MICROBIOLOGY AND CLASSIFICATION

A new third species of Chlamydia, called C pneumoniae, has been established for the TWAR organism. 24 We first thought that the new organism belonged to the C psittaci species because it was clearly not a C trachomatis. 2 •8 Subsequent studies showed that the organism was unique from both currently recognized species. 8 ·10•11 •17 •19 Justification for establishing a new species rests primarily on studies of the ultrastructure of the elementary body 10 and DNA analysis. 11 ·19 The organism also is serologically unique from the C trachomatis species and all C psittaci strains tested. 2 •8 Ultrastructural studies have shown that C pneumoniae has a pear-shaped elementary body. The cytoplasmic mass is round and there is a large periplasmic space. This is entirely different from the elementary bodies of C trachomatis and C psittaci that are round, dense, and have a narrow or barely discernible periplasmic space. 10 Genetic analysis revealed 10 percent or less DNA relatedness between C pneumoniae and the other two species, while homology among the TWAR strains tested was 94 percent or greater. 19 The restriction endonuclease patterns were similar among C pneumoniae isolates and readily distinguishable from C trachomatis and C psittaci strains. 11 The C pneumoniae also can be differentiated from the other species by using cloned C pneumoniae restriction fragments or whole chromosomal DNA as probes. 11 *From The School of Public Health and Community Medicine, the University of Washington, Seattle. Supported in part by USPHS grant AI-21885 from the National Institutes of Health. Studies by the author reviewed in this paper that involved human subjects utilized informed consent and were approved by the University ofWashingtons Human Subjects Committee. Reprint requests: Dr. Grayston, Epidemiology, SC-36, University of Washington, Seattle 98195

664

Table 1 shows characteristics of the C pneumoniae organism compared with the other species. All TWAR isolates so far tested appear identical in serologic findings, ultrastructural morphologic studies, and by DNA analysis. Therefore, the TWAR organism is the only strain or serovar of C pneumoniae so far recognized. The name TWAR has no specific meaning; it is a strain name from the laboratory designation of the first two isolates, TW-183 + AR-39. LABORATORY DIAGNOSIS

Laboratory diagnosis is based on isolation of the organism and serologic findings. Isolation has been difficult. The TWAR organisms grow poorly by current methods, or they would have been discovered earlier. Our method for isolation is similar to the procedure we use for C trachomatis in HeLa 229 cell culture 27 modified for C pneumoniae. 8 •16•20 The inclusions are smaller than seen with other Chlamydia and they are slow to maturate. McCoy cells are not as sensitive as He La 229 cells. 8 Many TWAR isolates fail to become adapted to HeLa 229 cells even after a number of passages. Two of our 28 isolates were obtained only in the yolk sac of embryonated chicken eggs. Adaptation to cell culture has been assisted by one or two passages in egg yolk sac. 8 With the exception of TW-183 that was isolated from the conjunctiva, all C pneumoniae isolates have been made from pharyngeal swab specimens. These specimens may not be ideal for isolation from patients with pneumonia or bronchitis. Specimens for isolation should be placed in Chlamydia SPG or 2SP transport media. They may be kept at 4°C if inoculated within 24 h. Slow freezing is better than rapid freezing. Specimens should be cooled at 4°C for one to four hours prior to freezing at

-7ooc_l6 Isolation has been greatly facilitated by the development of a TWAR monoclonal antibody which conjugated to fluorescein is used to identify inclusions in cell culture. 2 A genus monoclonal antibody will also stain the inclusions but does not identify them as C pneumoniae. Two serologic tests are useful in diagnosis. The Chlamydia pneumoniae, Strain TWAR (J. Thomas Grayston)

Table 1-Some Characteristics of the Three Species ofChlamydia Species

Characteristics

C pneumoniae

c psittaci

C traclwmotis Trachoma/LGV

Natural hosts DNA homology %-ref to C pneumoniae'• Mol% G+C of DNA'" Plasmid" No. of serovars"" Morphology ofEB 10 Morphology of inclusion• Glycogen in inclusion (2) Folate biosynthesis (14) Lethal by IC in mice (8) Folicular conjunctivitis in monkey (6) Cell culture enhancement by centrifugation (16)

Humans 100 40 No 1 PeW"shaped Oval, dense No No No No Yes

Birds, lower mammals 10 41 Yes (rare no) Unknown Round Variable, dense No No Yes No No

Humans 10 41142 Yes 1213 Round Oval, vacuolar Yes Yes No/yes Yes/no Yes/no

micro-IF test with TWAR antigen is specific for C pneumoniae and the complement fixation test measures antibodies against all Chlamydia. Table 2 shows the interpretation of results with these two tests. The micro-IF test can distinguish between antibodies in the IgM and IgG serum fractions. This is very useful in determining current or recent infections vs previous infection. Antibody in the IgG serum fraction without titer rise is interpreted as indicating previous infection with the organism. This antibody is used in population antibody prevalence surveys. The micro-IF test developed for C trachomatisZB remains the only sensitive and specific serologic test for Chlamydia. The test requires careful microscopy to avoid reading nonspecific (genus) reactions. While the test is widely used in chlamydia! research, only a few commercial laboratories are doing this test for TWAR antibody. The acute antibody response, indicating current or recent C pneumoniae infection, appears in two different patterns. Knowledge of these patterns is important in interpreting serologic studies. One pattern is associated with first or primary infection and one with reinfection. In first infection, a prompt Chlamydia CF antibody response is seen. The TWAR micro-IF antibody in the IgM serum fraction appears about three weeks after the onset of illness and antibody in the IgG serum fraction does not appear until six to eight Table 2-Serologic Tests for TWAR, with Definition of

Positive Results

Microimmunofluorescence with TWAR antigen Acute antibody (current infection): 4-fold titer rise lgM~1:16

IgG~1:512

Preexisting Antibody (previous infection): Chlamydia CF (not specific):

IgG ~1:16 and <1:512 4-fold titer rise ~1:64

weeks after onset. In reinfection there is often no CF antibody and no IgM antibody (or they may be in low titer). The TWAR IgG antibody often appears within one to two weeks and may reach very high titer. Because of the unusually slow development of microIF antibody in first infection, convalescence blood specimens should be obtained no earlier than three weeks after onset. The absence of CF and IgM antibody response in reinfection may make serologic diagnosis difficult. The use of the CF test is limited by the fact that it frequently fails to show antibody in reinfection. Most older patients with TWAR disease will not show a CF antibody response. Only a few patients with evidence of TWAR infections in our hospitalized pneumonia studies have had CF antibody. 9•23•26 Conversely, a rise in Chlamydia CF antibody titer may more often indicate C pneumoniae than C psittaci infection, because the former is much more frequent, unless the patient has been exposed to a sick bird or has occupational exposure. If laboratory diagnosis of TWAR infection is to become generally available, more sensitive methods for growth of the organism and easier serologic tests are needed. CLINICAL DISEASES

A summary of current knowledge of the clinical diseases associated with TWAR infection has been prepared based on a series of studies, some not yet published in their entirety, listed in Table 3. Table 3 shows that while relatively few cases of C pneumoniae infection have been identified in studies of endemic infection in the United States and Canada, larger numbers (a much higher percentage of those studied) have been identified in the Scandinavian epidemics. Tables 4 to 7 summarize the clinical diseases at different ages. The best information on incidence is in young adults (Table 4). While there is considerable CHEST I 95 I 3 I MARCH, 1989

885

Table 3-Liat of studies ofC pneumoniae, Strain TWAB, Infections No. Persons Studied Primary care clinics UW Students-1983-87 Group Health-1985-87 Hospitalized pneumonia Halifax -1983-85 Pittsburgh -1986-87 Seattle-1980-81 Pneumonia in HMO Group Health, Seattle-1963-75 Scandinavian epidemics Norway, Sweden, Denmark-1981-84 Military company, Denmark-1976 Als, Denmark-1979 Kajaani, Finland -1978 Military trainees, Finland -1977-87 Special groups Filipino children <5 yr old with lower respiratory tract infection Sarcoidosis Chronic obstructive pulmonary disease Endocarditis Myocarditis

No. TWAR Infections

Reference

647 784

20 26

2,3,22* 3,22*

301 359 198

18 27 20

9 26* 23

1,082

66

22*

1,102 14 25 34 234

520 7 21 32 137

5* 5 5

203 22 79

16 19 2 1 3

NA

7,13,18* 21 12 25

Unpublished data Unpublished data

*More to be published.

variation from year to year, about 10 percent of pneumonia cases have been shown to be associated with TWAR infection. 3 •22 These pneumonias have been mild with short-term disability, but if they are not treated with tetracycline, or erythromycin, there may be prolonged symptoms with malaise and cough extending for several months. The TWAR infection has been demonstrated in about 5 percent of both bronchitis and sinusitis cases. 3 •22 The sinusitis often is associated with lower respiratory tract symptoms. The TWAR infection has been found in only 0.5 to 1 percent of patients with primary pharyngitis. However, pharyngitis is an important and frequent symptom in lower respiratory tract TWAR infections. Current data suggest that seven of ten TWAR infections may be asymptomatic or only mildly symptomatic. 13•18 In outpatients, the clinical characteristics of the atypical pneumonia caused by C pneumoniae were in general similar to the disease caused by Mycoplasma pneumoniae. 2 A characteristic of TWAR pneumonia Table 4-Summary of Clinical DiBetJBe with C pneumoniae Infection in 'leen-agers and Young Adults Mostly First TWAR Infection Pneumonia-mild, may be prolonged Bronchitis-often prolonged Sinusitis-primary or with above Pharyngitis-primary Pharyngitis occurs in more than 50% ofTWAR Pneumonia and bronchitis, many biphasic Asymptomatic or nonfebrile upper respiratory tract infection

%TWAR 10

5 5

(7 of 10)

and bronchitis is the frequent occurrence of relatively severe sore throat often with hoarseness. In addition, a biphasic illness frequently has been observed. This consists of a pharyngitis illness often resulting in a clinic visit. Following a negative throat culture, the patient is treated symptomatically and improves. One to three weeks later there is onset of pneumonia or bronchitis. Table 5 lists TWAR infections in adults. Their infection may be either a first infection or a reinfection. Pneumonia and bronchitis remain the most common syndromes associated with TWAR infection. Pharyngitis and sinusitis often accompany them. A febrile illness without localizing signs has been associated with TWAR infection. In addition, a few cases of sarcoidosis, myocarditis and endocarditis have had associated TWAR infection shown serologically. 12 Other Chlamydia also have caused myocarditis and endocarditis. The etiologic role of TWAR in these syndromes remains to be proven. While no deaths have yet been attributed to uncomplicated TWAR pneumonia, severe illness has been Table 5-Summary of Clinical Disease with C pneumoniae infection in Adults Both First and Reinfection TWAR Disease Pneumonia-mild to severe Bronchitis-often prolonged Pharyngitis-often accompanies above Sinusitis-may accompany above Febrile illness-"influenza-like" Asymptomatic-common Sarcoidosis, myocarditis, endocarditis-(?)

Chlamydia pneumoniae, Strain TWAR (J. Thomas G18Y5ton)

Table 6- Summary of Clinical Diseases with C pneumoniae Infections in Older Adult8 Reinfection 1WAR Disease Pneumonia-often complicated and severe (some deaths) in persons with chronic disease, eg, chronic obstructive pulmonary disease, cardiac failure Bronchitis-may be chronic Sinusitis-may be chronic

observed in otherwise healthy adults. These illnesses have been similar to severe psittacosis infections, and only vigorous antibiotic and supportive therapy have prevented death. 3 Table 6 emphasizes that TWAR infections in older adults are usually reinfections and that the syndromes caused often are chronic. When TWAR pneumonia occurs in older adults with preexisting illnesses, such as chronic obstructive pulmonary disease or cardiac failure, the illness may be severe and occasionally result in death. Hospitalized patients with pneumonia that have been shown to have TWAR infection have not had clinical or laboratory findings that differ from others in the same study. 9 ·23 •26 Less is known about TWAR infection in children (Table 7). Population antibody prevalence studies suggest that frequent infection is occurring between 8 and 15 years of age. The TWAR illnesses in children have been similar to those found in teen-agers and young adults. Only a few children less than five years of age have been shown to have TWAR infections in the Scandinavian studies and none has been found in Seattle. However, in the Philippines in children less than five years of age, a number of cases of severe lower respiratory tract infection with one death have been associated with TWAR infection. 21 Clinical laboratory findings in TWAR infections are not remarkable. The white blood cell count is usually normal and the sedimentation rate is elevated. Chest radiographs in TWAR pneumonia characteristically show single lesions that are subsegmental and may be in any part of the lung. Consolidation is rarely seen. In hospitalized pneumonia patients with TWAR infection a few have had bilateral lesions. These patients hav~ had pleurisy more frequently than comparison patients. 9 •23 Table 7 -Summary of Clinical Disease with C pneumoniae lnfoctions in Children First 1WAR Infection 6-12 years of age Similar to teen-age, can be milder with more asymptomatic infections 1-5 years of age In United States and Scandinavia, uncommon; in tropical countries, may cause acute serious lower respiratory tract infection

Apparent hospital-acquired pneumonia has been observed in patients admitted with severe trauma or undergoing major surgery. The mechanism of infection in the hospital has not been identified, but all the patients were intubated and had some surgical procedure. It is possible that the cases of nosocomial pneumonia seen in patients with serious illnesses may represent endogenous reinfection or reactivation of the TWAR organism in the lung rather than human transmitted infection in the hospital. 23 The role of immunopathologic findings in clinical diseases caused by TWAR infection has not yet been determined. Some of the most severe TWAR infections have been seen in older persons who were probably undergoing reinfection. Reinfection TWAR disease has been demonstrated in military trainees. 18·22 Both milder and more severe illnesses were seen with reinfection than with first infections. One patient undergoing reinfection developed manifestations of sarcoidosis. Immunopathologic findings have been well established as playing an important role in the diseases caused by C trachomatis. Fully developed trachoma with conjunctival scarring and pannus formation follows reinfection. 29 Infertility and ectopic pregnancy due to pathologic changes of the fallopian tubes have been demonstrated experimentally to be caused by reinfection. 30 The Fitz-Hugh-Curtis syndrome of perihepatitis occurred in women with evidence of reinfection with C trachomatis. 31 ANTIBIOTIC SENSITIVfiY AND TREATMENT

Clinical experience has shown that small doses or short courses of erythromycin for TWAR pneumonia have frequently been followed by continuing symptoms with malaise and cough for several months. 2 A completely different micro-IF IgG antibody response was shown in patients treated with penicillin or ampicillin compared with tetracycline. High-titer IgG antibody developed after treatment with penicillinampicillin but that antibody response was depressed or absent in patients promptly treated with tetracycline.13 We have published laboratory data on antibiotic and sulfa drug sensitivities of C pneumoniae . 14 Tetracycline and erythromycin are the most effective drugs as they are with other Chlamydia. Sulfa is not effective against C pneumoniae. The same is true with most C psittaci strains, but all C trachomatis strains are sensitive to sulfa drugs. While both clinical experience and laboratory studies have shown that tetracycline and erythromycin drugs are effective against TWAR organisms, intensive prolonged therapy is recommended. Until careful comparative clinical studies are performed, our current recommendation for treatment is similar to that recommended for C psittaci infection: 2 glday of CHEST I 95 I 3 I MARCH, 1989

667

tetracycline or e r y t h r o m y c i n for ten days to two weeks.

infectiousness of a person with T W A R infection. No

An alternate therapy schedule can b e 1 g/day for 21

seasonability has b e e n demonstrated for C

days. T h e clinician should not be surprised if the

niae infection. I m m u n i t y following infection with all Chlamydia

patient requires additional antibiotic therapy.

of C pneumoniae

E a r l y in our studies of T W A R , we recognized that antibody

indicating

previous

has b e e n popr. T h e role of reinfection,

both exogenous and endogenous, in the epidemiology

EPIDEMIOLOGY

TWAR

pneumo-

infection

was

will b e important to elucidate.

T h e population antibody studies that have b e e n carried out suggest that T W A R antibody is m o r e

found frequently in different populations, much m o r e

c o m m o n in tropical countries (Panama, Taiwan) and

frequently than C trachomatis

less c o m m o n in m o r e n o r t h e r n countries (Canada,

antibody. 4 This anti-

body in the IgG serum fraction was found in about 5 0

Scandinavia). T h e findings from the study of Filipino

p e r c e n t of adults in seven different countries from

children 2 1 support the possibility that T W A R infections

around the world. It varied from around 4 0 p e r c e n t in

are occurring at an earlier age and m o r e frequently in

Nova Scotia to m o r e than 6 0 p e r c e n t in Taiwan and

tropical countries.

Panama. In each area m o r e antibody was found in m e n than women.

W h i l e no explanation yet exists for the epidemics that have o c c u r r e d in Scandinavia c o m p a r e d with

W e have tested enough s e r u m from all ages in

e n d e m i c infections in the United States, it may be

Seattle and D e n m a r k to d e t e r m i n e prevalence by age.

that fewer infections in m o r e n o r t h e r n climates allow

T h e antibody rates in both areas are very low in young

the buildup of adequate n u m b e r s of susceptible people

children ( < 5 p e r c e n t up to five years of age and < 1 0

to sustain large outbreaks.

p e r c e n t to ten years of age) and increase rapidly in

Investigators at the Institute of Ophthalmology in

teen-agers (30 to 4 0 percent). T h e rates continue to

L o n d o n have r e p o r t e d isolation of a Chlamydia strain

increase into middle age (50 percent) and remain high

said to r e s e m b l e T W - 1 8 3 . 3 2 T h e y used this strain as

in old age (50 percent).

antigen

T h e persistence of T W A R antibody in old age, a

in the microimmunofluorescence

test and

studied sera from England and a n u m b e r of other

where

countries. T h e antibody prevalence in the various

antibody prevalence falls off sharply after ages 4 0 to

populations w e r e similar to those discussed above

phenomenon 5 0 years,

not seen with C trachomatis

suggests that reinfection with T W A R

is

c o m m o n . In view of the e x p e c t e d decay of antibody

except

that

they

showed

more

variability

in

the

f r e q u e n c y of antibody by country. 3 3

after infection, the finding of T W A R antibody in 5 0 p e r c e n t of the adult population at one t i m e suggests that most people are infected and reinfected during their life. T h e lack of antibody in young children and the higher antibody

rates

in adult m e n

than

women

suggests that the family unit is not the primary place for T W A R transmission as it is for many viral respiratory infections and for M pneumoniae.

Transmission

in schools and other institutions, as well as in military training is suggested both by the known epidemics and by the curve of population antibody prevalence. This does not mean that transmission in families does not occur. W e have d e m o n s t r a t e d clusters of T W A R infections in families serologically. Careful studies of the c i r c u m s c r i b e d T W A R e p i d e m ics in Finland failed to reveal any e v i d e n c e of a bird or animal reservoir for the organism. 1 - 7 1 3 T h e very high worldwide infection rates shown by serologic studies would argue against such reservoirs

being

important in transmission o f the organism. All evid e n c e suggests that T W A R is a primary h u m a n pathogen without a bird or animal reservoir that is transmitted from human to human. However, the m o d e and place of transmission and the incubation period are unknown. T h e r e is no information yet on a carrier status for the organism or factors that play a role in 668

ACKNOWLEDGMENTS: The author wishes to acknowledge the contribution to information about TWAR of his principal colleagues: L. A. Campbell, C. C. Kuo, C. H. Mordhorst, P. Saikku and S. P Wang. REFERENCES 1 Saikku P, Wang SP, Kleemola M, Brander E, Rusanen E, Grayston JT. An epidemic of mild pneumonia due to an unusual Chlamydia psittaci strain. J Infect Dis 1985; 151:832-39 2 Grayston JT, Kuo CC, Wang SP, Altman J. A new Chlamydia psittaci strain called TWAR from acute respiratory tract infections. N Engl J Med 1986; 315:161-68 3 Grayston JT, Kuo CC, Wang SP, Cooney MK, Altman J, Marrie TJ, et al. Clinical findings in TWAR respiratory tract infections. In: Oriel JD, et al, eds. Chlamydial infections. Cambridge, England: Cambridge University Press, 1986;337-40 4 Wang SP, Grayston JT. Microimmunofluorescence serological studies with the TWAR organism. In: Oriel JD, et al, eds. Chlamydial infections. Cambridge, England: Cambridge University Press, 1986;329-32 5 Mordhorst CH, Wang SP, Grayston JT. Epidemic 'ornithosis' and TWAR infection, Denmark 1976-85. In: Oriel JD, et al, eds. Chlamydial infections. Cambridge, England: Cambridge University Press, 1986;325-28 6 Kuo CC, Chen HH, Wang SP, Grayston JT. Characterization of TWAR strains: a new group of Chlamydia psittaci. In: Oriel JD, et al, eds. Chlamydia infections. Cambridge, England: Cambridge University Press, 1986;321-24 7 Saikku P, Visakorpi R, Kleemola M, Wang SP, Grayston JT. Chlamydia psittaci TWAR-strains as a frequent cause of military pneumonia epidemics in Finland. In: Oriel JD, et al, eds. Chlamydia pneumoniae, Strain TWAR (J. Thomas Grayston)

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