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(Chest. 2001;119:1416-1419.)
© 2001 American College of Chest Physicians

Prevalence of Asymptomatic Infection With Chlamydia pneumoniae in Subjectively Healthy Adults*

Naoyuki Miyashita, MD, PhD; Yoshihito Niki, MD, PhD, FCCP; Masamitsu Nakajima, MD, PhD, FCCP; Hiroshi Fukano, MD and Toshiharu Matsushima, MD, PhD, FCCP

* From the Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki City, Okayama, Japan.

Correspondence to: Naoyuki Miyashita, MD, PhD, Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki City, Okayama 701-0192, Japan; e-mail: nao{at}med.kawasaki-m.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: To investigate the prevalence of asymptomatic infection with Chlamydia pneumoniae in subjectively healthy adults.

Design: Prospective study.

Setting: Kawasaki Medical School Hospital in Kurashiki, Japan.

Participants: Total of 1,018 asymptomatic healthy adults (age range, 22 to 50 years; mean age, 32.4 years; 482 men and 536 women).

Measurements and results: Nasopharyngeal swab specimens were obtained from all subjects and analyzed by isolation in cell cultures and polymerase chain reaction (PCR) test for C pneumoniae. Serum samples were also obtained and tested for C pneumoniae-specific antibodies by the microimmunofluorescence test. Of 1,018 specimens tested, 14 specimens (1.4%) were judged positive by culture and/or the PCR. Thirteen specimens were PCR positive, and 4 specimens were culture positive. There were three specimens positive by both tests and 11 specimens positive with discrepancies in culture and PCR results. None of the individuals met the serologic criteria for acute infection. Of 1,018 sera tested, 64.1% of men and 58.0% of women had antibody to C pneumoniae. The overall prevalence of antibody was 60.9%. Forty individuals (3.9%) had an IgG titer of >= 1:512 or IgM titer of >= 1:16.

Conclusions: Our data suggest that asymptomatic infection with C pneumoniae may occur in subjectively healthy adults.

Key Words: asymptomatic infection • Chlamydia pneumoniae • healthy adults • persistent infection


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C;-2qhlamydia pneumoniae has been established as an important cause of acute respiratory illnesses, including pneumonia, bronchitis, pharyngitis, and sinusitis. Approximately 10% of cases of community-acquired pneumonia are associated with C pneumoniae.1 2 Since seroepidemiologic studies have demonstrated that 50 to 70% of adults have antibody to C pneumoniae, it is estimated that nearly everyone acquires at least one C pneumoniae infection during their lifetime.2 In addition, many studies2 3 4 5 6 7 8 have suggested that there is a direct association between C pneumoniae infection and other clinical manifestations, such as atherosclerotic cardiovascular disorders, acute exacerbations of COPD, and asthma.

According to some studies9 10 11 12 13 in Western countries, asymptomatic infections may be common. Isolations and positive polymerase chain reaction (PCR) findings without any respiratory symptoms may be a sign of an asymptomatic nasopharyngeal infection with C pneumoniae. We encountered seven subjects with asymptomatic C pneumoniae infection who were exposed to patients with C pneumoniae respiratory tract infection at a junior high school during an outbreak of C pneumoniae.14 However, there have been no large-sample-size prospective studies of asymptomatic infection with C pneumoniae in Asia.

In this study, we prospectively investigated the prevalence of asymptomatic infection with C pneumoniae in subjectively healthy Japanese adults by isolation and PCR from nasopharyngeal swabs and by serology.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
The subjects in the study population were asymptomatic (inclusive age range, 22 to 50 years [n = 1,018]; age range, 22 to 30 years [n = 484]; age range, 31 to 40 years [n = 356]; age range, 41 to 50 years, [n = 178]; mean, 32.4 years; 482 men and 536 women) and included medical students and staff at Kawasaki Medical School Hospital between April 1994 and December 1999. Subjects were excluded from the study if they reported a history of infection with C pneumoniae, if they had received antibiotics within 2 weeks preceding enrollment, or if they reported having had a clinical syndrome compatible with pharyngitis, sinusitis, bronchitis, or pneumonia within the preceding 3 months before enrollment. Informed consent was obtained from all subjects.

Culture and PCR
Nasopharyngeal swab specimens were obtained from all subjects for isolation in cell cultures and the PCR. The swab specimens were placed in a sucrose-phosphate-glutamate transport medium. Culturing for C pneumoniae was performed in cycloheximide-treated HEp-2 cells grown in a 24-well cell culture plate as reported previously.15 All specimens were passed twice. Culture confirmation was done by fluorescent-antibody staining with C pneumoniae species-specific monoclonal antibody.15 16

The C pneumoniae-specific primers used for PCR were from the DNA base sequence within the 53-kd protein gene established in our laboratory.17 This assay was performed as previously described, and it was carried out without prior knowledge of the culture results. The cell-culture-grown C pneumoniae strain KKp-15 was used as the positive control,18 and a chlamydia transport medium was used as the negative control in every run. After electrophoresis of amplification products on a 1.5% agarose gel at 100 V, the band was visualized by staining with ethidium bromide. The appearance of a 499 base pair amplification product was taken as positive. The sensitivity and specificity of our PCR assay were 91.7% and 93.6%, respectively, when compared with the culture,14 and they were 92.9% and 99.2%, respectively, when we compared them with another primer sets, HL-1 and HR-1, reported by Campbell et al.19

Serology
Serum samples were obtained from all subjects and stored at - 70°C until testing. The microimmunofluorescence test was used for titration of IgG and IgM antibodies against C pneumoniae,1 2 using formalinized elementary bodies of the C pneumoniae KK-pn15 strain as antigens.18 Rheumatoid factors were absorbed with Gullsorb (Gull Laboratories; Salt Lake City, UT) before IgM titrations. The serologic criterion for a positive test result was a titer >= 1:16 for IgG or IgM.1 2

If available, follow-up nasopharyngeal swab specimens and sera were obtained from subjects with positive findings by culturing and/or PCR. Asymptomatic infection with C pneumoniae was defined as isolations and/or positive PCR findings without any respiratory symptoms.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of 1,018 specimens tested, 14 specimens (1.4%) tested positive by either culture and/or PCR (Table 1 ). Thirteen specimens were PCR positive and 4 specimens were culture positive. There were three specimens positive by both tests and 11 specimens positive with discrepancies in the culture and PCR results; 1 specimen was culture positive and PCR negative, and 10 specimens were culture negative and PCR positive. The sensitivity of the PCR was 75.0% and the specificity was 99.0%. There were differences in the prevalence rates of C pneumoniae infection in specimens taken by year, 0.7% in 1994, 1.1% in 1995, 1.8% in 1996, 0% in 1997, 1.0% in 1998, and 3.2% in 1999 (Table 2 ). In all the individuals with positive culture findings, very few (from two to six) C pneumonaie inclusions were found in the second passage of the cell culture, which may indicate the low numbers of organisms present in asymptomatic infections. Of the 14 individuals with positive findings by either culturing and/or the PCR, 8 individuals (57.1%) had preexisting antibody and 6 individuals (42.9%) had negative IgG antibody for C pneumoniae. No IgM antibody titers of C pneumoniae were found in these individuals. Hence, the prevalence of asymptomatic nasopharyngeal infection in our study group was 1.4%.


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  		Table 1. Culture, PCR, and Serologic Results for Specimens Positive for C pneumoniae by Culture and/or PCR*

 

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  		Table 2. Prevalence Rates of C pneumoniae Infection in Each Year

 
Serologic results are shown in Figure 1 . Of 1,018 sera tested, 64.1% of men and 58.0% of women had antibody to C pneumoniae. The overall prevalence of antibody was 60.9%, and it increased with age. Forty individuals (3.9%) had a IgG titer of >= 1:512 or IgM titer of >= 1:16. Thirty-six individuals had an IgG titer of >= 1:512 and 4 individuals had an IgM titer of >= 1:16.



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  		Figure 1. Prevalence of antibodies to C pneumoniae in 1,018 asymptomatic healthy subjects according to age.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Serologic studies of C pneumoniae infection have shown > 50% of adults worldwide to have serologic evidence of previous infection. In our area of Japan, a high prevalence of antibodies against C pneumonaie was also found among healthy persons.4 6 8 Gnarpe et al9 first examined asymptomatic C pneumoniae pharyngeal infection using cell cultures, and the prevalence of infection was determined to be 4.7% of healthy adults (11 of 234). Subsequently, Emre et al10 and Block et al11 detected the C pneumoniae organism by cell culture in 4.9% and 5.0% of asymptomatic healthy children, respectively. On the other hand, Gaydos et al12 and Hyman et al13 identified nasopharyngeal infection with C pneumoniae by culturing and/or the PCR in only 1 of 80 asymptomatic patients (1.2%) and 2 of 104 healthy adults (1.9%), respectively. In our large-sample-size study, we were able to detect C pneumonaie from nasopharyngeal specimens by culturing and/or the PCR in 14 of 1,018 subjectively healthy adults (1.4%). Our results are consistent with those reported by Gaydos et al12 and Hyman et al13 and indicate that asymptomatic upper respiratory tract infection due to C pneumoniae occurs in other healthy populations. However, the prevalence judged by our cell cultures was much lower than that reported by Gnarpe et al,9 Emre et al,10 and Block et al11 (0.4% vs 4.7 to 5.0%). The difference may be because of the use of a different cohort from a different geographic area than that employed in their study.

In a previous study,14 we examined seven individuals with asymptomatic C pneumoniae infection during a C pneumoniae epidemic. Of these seven individuals from whom follow-up cultures were obtained, the culture finding for only one individual remained positive at 2 months. This student developed an antibody response that was suggestive of acute infection. At that time, we found seven cases of C pneumoniae pneumonia.14 All seven patients with pneumonia were treated with antichlamydial antibiotics (eg, minocycline and clarithromycin) and showed good clinical responses, but two of these patients became asymptomatic carriers. In these patients, C pneumoniae was cultured persistently for 3 months and 5 months, respectively, after the resolution of symptoms. Furthermore, we have also seen persistent C pneumoniae infection for up to 2 years in a patient with diffuse panbronchiolitis who was asymptomatic most of the time but also had exacerbations.20 In this study, follow-up culture, PCR, and serology results were obtained from 10 of 14 individuals. In three individuals, C pneumoniae was detected persistently for 4 to 12 weeks, but they showed no serologic evidence of acute infection (fourfold increase in IgM or IgG titer). We believe that these 14 individuals who had evidence of infection during our study were asymptomatic carriers of C pneumoniae, and we suggest that these individuals are a reservoir for C pneumoniae within the community. Although the duration of asymptomatic infection could not be determined from the results of this study because of the small sample size of asymptomatic infection, further study seems warranted.

In serologic findings measured by the microimmunofluorescence test, Grayston et al1 and Kuo et al2 defined the criterion for acute C pneumoniae infection as IgM >= 1:16 or IgG >= 1:512 using single serum. Gaydos et al12 and Hyman et al13 reported a surprisingly high proportion of their patients (18.8% and 18.4%, respectively) met the serologic criteria for acute C pneumoniae infection. Similar results were also reported by Kern et al,21 who indicated that 12.9% (19 of 147) of healthy subjects were found to have serologic evidence of acute C pneumoniae infection. In our study, however, only 3.9% of healthy adults (40 of 1,018) had a titer of IgG >= 1:512 or IgM >= 1:16, contradictory to former reports.12 13 21 Such differences may have been due to a very high seroprevalence of antibody to C pneumoniae in their cohort, which implies a high incidence of previous infection compared to our cohort. These serologic results together with our data indicate that the definition of acute or current infection using IgG >= 1:512 is controversial. In conclusion, we suggest that asymptomatic infection with C pneumoniae may occur in subjectively healthy adults.


   Footnotes
 
Abbreviation: PCR = polymerase chain reaction

Received for publication January 26, 2000. Accepted for publication November 9, 2000.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grayston, JT, Campbell, LA, Kuo, CC, et al (1990) A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis 161,618-625[ISI][Medline]
  2. Kuo, CC, Jackson, LA, Campbell, LA, et al (1995) Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 8,451-461[Abstract]
  3. Saikku, P, Leinonen, M, Tenkanen, L, et al (1992) Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med 116,273-278
  4. Miyashita, N, Toyota, E, Sawayama, T, et al (1998) An association of an antibody against Chlamydia pneumoniae and coronary heart disease observed in Japan [letter]. Eur Heart J 19,971
  5. von Hertzen, L, Alakarppa, H, Koskinen, R, et al (1997) Chlamydia pneumoniae infection in patients with chronic obstructive pulumonary disease. Epidemiol Infect 118,155-164[CrossRef][Medline]
  6. Miyashita, N, Niki, Y, Nakajima, M, et al (1998) Chlamydia pneumoniae infection in patients with diffuse panbronchiolitis and COPD. Chest 114,969-971[Abstract/Free Full Text]
  7. Hahn, DL, Dodge, RW, Golubjatnikov, R (1991) Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA 266,225-230[Abstract]
  8. Miyashita, N, Kubota, Y, Nakajima, M, et al (1998) Chlamydia pneumoniae and exacerbations of asthma in adults. Ann Allergy Asthma Immunol 80,405-409[ISI][Medline]
  9. Gnarpe, J, Gnarpe, H, Sundelof, B (1991) Epidemic prevalence of Chlamydia pneumoniae in subjectively healthy persons. Scand J Infect Dis 23,387-388[ISI][Medline]
  10. Emre, U, Roblin, PM, Gelling, M, et al (1994) The association of Chlamydia pneumoniae infection and reactive airway disease in children. Arch Pediatr Adolesc Med 148,727-731[Abstract]
  11. Block, SL, Hammerschlag, MR, Hedrick, J, et al (1997) Chlamydia pneumoniae in acute otitis media. Pediatr Infect Dis J 16,858-862[CrossRef][ISI][Medline]
  12. Gaydos, CA, Roblin, PM, Hammerschlag, MR, et al (1994) Diagnostic utility of PCR-enzyme immunoassay, culture, and serology for detection of Chlamydia pneumoniae in symptomatic and asymptomatic patients. J Clin Microbiol 32,903-905[Abstract/Free Full Text]
  13. Hyman, CL, Roblin, PM, Gaydos, CA, et al (1995) Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction-enzyme immunoassay and culture. Clin Infect Dis 20,1174-1178[ISI][Medline]
  14. Soda, K, Kishimoto, T, Kubota, Y, et al (1997) An outbreak of Chlamydia pneumoniae in a junior high school and its district: presence of asymptomatic oropharyngeal carriers in healthy subjects and in cases of pneumonia after a clinical cure [in Japanese]. Amakusa Med J 11,17-28
  15. Miyashita, N, Matsumoto, A, Soejima, R, et al (1996) Evaluation of a direct fluorescent antibody assay for detection of Chlamydia pneumoniae. Jpn J Assoc Infect Dis 70,224-231
  16. Iijima, Y, Miyashita, N, Kishimoto, T, et al (1994) Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans. J Clin Microbiol 32,583-588[Abstract/Free Full Text]
  17. Kubota, Y (1996) A new primer pair for detection of Chlamydia pneumoniae by polymerase chain reaction. Microbiol Immunol 40,27-32[ISI][Medline]
  18. Miyashita, N, Kubota, Y, Kimura, M, et al (1994) Characterization of Chlamydia pneumoniae strain isolated from a 57-year-old man. Microbiol Immunol 38,857-864[ISI][Medline]
  19. Campbell, LA, Melgosa, MP, Hamilton, DJ, et al (1992) Detection of Chlamydia pneumoniae by polymerase chain reaction. J Clin Microbiol 30,434-439[Abstract/Free Full Text]
  20. Miyashita, N, Matsumoto, A, Kubota, Y, et al (1996) Continuous isolation and characterization of Chlamydia pneumoniae from a patient with diffuse panbronchilitis. Microbiol Immunol 40,547-552[ISI][Medline]
  21. Kern, DG, Neill, MA, Schachter, J (1993) A seroepidemiologic study of Chlamydia pneumoniae in Rhode Island. Chest 104,208-213[Abstract/Free Full Text]



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