Outbreak of Respiratory Tract Infections on an Islet in Korea: Possible Chlamydia pneumoniae Infection

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1 Jpn. J. Infect. Dis., 59, , 2006 Original Article Outbreak of Respiratory Tract Infections on an Islet in Korea: Possible Chlamydia pneumoniae Infection Kwang-Jun Lee, Su-Jin Kwon, Bo-Ram Choi, Song-Mee Bae, Toshio Kishimoto 1, Shuji Ando 1, Chikako Mashida 2, Young-Hee Lee, Hee-Bok Oh and Ki-Sang Kim* Division of Bacterial Respiratory Infections, Infectious Disease Research Center, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul , Korea; 1 Laboratory of Rickettsia and Chlamydia, Department of Virology I, National Institute of Infectious Diseases, Tokyo ; and 2 Pharmaceutical Division, Hitachi Chemical Company, Ibaraki , Japan (Received September 30, Accepted July 7, 2006) SUMMARY: In March 2004, we experienced an outbreak of Chlamydia pneumoniae infection on an islet of Korea. In order to assess the significance of the epidemic, we performed a mass examination of 137 students (7-16 years old; male, 69; female, 58) at a school. The examination consisted of a questionnaire inquiring about respiratory symptoms, a serum antibody test for C. pneumoniae using a microimmunofluorescence (MIF) method and enzyme-linked immunosorbent assay (ELISA), and nasopharyngeal swab tests to detect of the organism by specific PCR and cell culture. The results demonstrated that 72 (58.3%) of the students had respiratory symptoms such as rhinorrhea, a sore throat, and/or cough or fever. The PCR positivity of acute-phase patients was 63% (12/19) and PCR positivity using the culture sample was 94% (18/19). However, the existence of the organism was not confirmed fluorescein isothiocyanate (FITC). ELISA, one of the serological methods utilized, demonstrated, in the same patients, 48% (13/27) positive IgM antibodies at the acute phase of the outbreak, and 16% (3/19) positive IgM antibodies during the convalescent phase. The index value (ID) 3.0 for single-sera IgG was 19% (5/27) and that for was 4% (1/27) at the acute phase; the corresponding percentages in the convalescent phase were 11% (2/19) and 5% (1/19), respectively. However, as regards paired sera, no patient demonstrated a 1.35 ELISA ID value at 2 weeks, or an increased value of 1.0 at 8 weeks after the onset of the outbreak. In the MIF experiment, the percent positivity of unpaired IgM from the acute phase was 58% (11/19). At the convalescent phase, this percentage was 47% (9/19); however, the positivity of paired serum IgG was 26% (5/19). In the same sample, the percentage of positive cases demonstrated by both ELISA and MIF approaches for single IgM was 37% (7/19) at the acute phase and 11% (2/19) at the convalescent phase. We were unable to isolate C. pneumoniae by cell culture, but we did obtain sufficient serological and PCR data to consider C. pneumoniae as the causative agent of the outbreak. Meaningful results were acquired in terms of serology, and were compared to the healthy population in Korea. Although it remains necessary to investigate the possibility of co-infection and to determine whether or not this outbreak coincides with the prevalence of influenza, it was unequivocally concluded that this outbreak of C. pneumoniae infection has occurred on an islet of Korea. INTRODUCTION Chlamydia pneumoniae is a new species of obligate intracellular bacteria that has recently been recognized as a third species of the genus Chlamydia. The organism was first isolated in 1965 from the conjunctiva of a Taiwanese child who was participating in a trachoma vaccine trial (1). C. pneumoniae causes several acute respiratory diseases, including pneumonia, bronchitis, sinusitis, and pharyngitis. C. pneumoniae is responsible for approximately 10% of the total number of cases of community-acquired pneumonia and 5% of the cases of bronchitis and sinusitis (2,3,18). The clinical symptoms produced by C. pneumoniae pulmonary infections are similar to those caused by other respiratory pathogens, with the addition of a few distinguishing features (4). *Corresponding author: Mailing address: Division of Bacterial Respiratory Infections, Infectious Disease Research Center, National Institute of Health, Korea Centers for Disease Control and Prevention, 5 Nokbun-Dong, Eunpyung-Ku, Seoul , Korea. Tel: , Fax: , kisangkim@nih.go.kr Swabs of the oropharynx can typically be used to obtain specimens for isolation. However, satisfactory techniques for isolation from sputum samples have not yet been developed. In addition, cell culturing has traditionally been the gold standard for obtaining a chlamydial diagnosis, but this approach is time consuming, technically demanding, and the improper collection and transportation of specimens may give false negatives. Therefore, the diagnosis of a chlamydial infection is typically made by either serological or PCR testing, both of which can be utilized for the prompt processing of clinical specimens. Specific serological tests in this context include enzyme-linked immunosorbent assay (ELISA), microimmunofluorescence (MIF) method, as well as other tests. To date, no epidemiological research has been conducted in Korea. The detection of C. pneumoniae-specific antibodies in human serum was the key factor leading to the discovery of this species of Chlamydia in the 1980s (1); at that time, C. pneumoniae was associated with pneumonitis (5,6). Since the identification of C. pneumoniae and the development of an MIF test for antibody analysis (7,8,19), several seroepidemiological studies have been conducted in order to increase our epidemiological and immunological understanding of C. 294

2 pneumoniae infections. The MIF assay recognizes conformational epitopes on intact chlamydial elementary bodies (EBs), and it is generally considered to be species-specific; currently, the MIF remains the gold standard for C. pneumoniae serology (9). However, utilization of the MIF method requires a special skill set, and there are significant differences among tests performed in different laboratories in terms of their relative sensitivity. A new technique for the detection of antibodies to C. pneumoniae, i.e., an ELISA approach, has recently been developed (11,12). We encountered an outbreak of C. pneumoniae infection on an islet of Korea in March The total population was 1,700 on the islet; and the number of elementary and junior high school students tested was 137. Since January 1, 2004, students became ill with symptoms that included fever, coughing, and sore throat. The clinical samples were tested for bacterial pathogens associated with similar symptoms, and we confirmed the presence of Chlamydia in patients tested who had also acquired a high fever with a concurrent cough and sore throat. A diagnosis of acute C. pneumoniae infection was based on the results of the PCR analysis, and anti-c. pneumoniae antibodies were measured by ELISA. MATERIALS AND METHODS Study sample: In March 2004, elementary and junior high school students who developed a high fever accompanied by a cough and sore throat, were tested for infection with C. pneumoniae. Of the total population, 137 subjects were elementary school (7-12 years old) and junior high school (13-16 years old) students. Fifty-seven percent of the elementary school and 60% of the junior high students developed symptoms. Chronic obstructive pulmonary disease (COPD) patients and the patients on the islet were diagnosed if they exhibited respiratory symptoms for more than 3 weeks. The normal subjects lived in Seoul and on the main island; these controls did not exhibit any respiratory or related symptoms for a sustained 3-week period. The controls were approximately the same age as the COPD patients and the patients living on the islet. Nasopharyngeal swabs and blood samples were obtained 1 week after the initial outbreak of symptoms; serum samples were taken 8 weeks later. Cell cultures and isolation of C. pneumoniae: Cell cultures: The host cell line, HEp-2, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va., USA). The cells were cultivated in Eagle s minimal essential medium (MEM) supplemented with 10% fetal bovine serum at 37 C in a 5% CO 2 atmosphere. Clinical specimens: Nasopharyngeal swab specimens were placed into 1.5 ml of sucrose-phosphate-glutamate buffer (ph 7.4) supplemented with gentamycin (40 g/ml), kanamycin (20 g/ml), streptomycin (20 g/ml), and amphotricin B (2.5 g/ml). Prior to storage at 75 C, a 600- L aliquot of the patient specimens was withdrawn for PCR analysis. Isolation in cell culture and inclusion-forming unit (IFU) assay: Patient specimens were shaken in suspension. Aliquots (200 L) of each sample were inoculated in duplicate onto HEp-2 cells (ATCC) grown on coverslips in shell viral. The shell viral was centrifuged at 3,000 g at room temperature for 1 h. The inoculum was replaced by 1 ml of Eagle s MEM supplemented with 10% fetal bovine serum, gentamycin (40 g/ml), kanamycin (20 g/ml), streptomycin (20 g/ml), amphotricin B (2.5 g/ml), and cyclohexamide (1 g/ml). The cells were incubated at 35 C in 5% CO 2 for 72 h, and the coverslips were fixed with methanol and stained for chlamydial inclusions with a fluorescein-conjugated genusspecific antibody (Seiken, Tokyo, Japan) to the Chlamydia lipopolysaccharide (LPS). Polymerase chain reaction (PCR) analysis: Extraction of DNA: DNA was extracted from C. pneumoniae in throat swabs using the PUREGENE genomic DNA Purification Kit (Gentra, Minneapolis, Minn., USA) procedure. C. pneumoniaeinfected cells were incubated in cell lysis solution with proteinase K (20 mg/ml) at 56 C overnight. Extracted DNA was stored at 20 C. PCR: Separate assays, targeting the 53-kDa cloned gene fragment, were used for the detection of C. pneumoniae (13). For the PCR analysis, which employed 53-1 and 53-2 with the nested primer pair 53-3 and 53-4, both amplification rounds utilized a 10-pM concentration of each primer and 1U of Taq Gold DNA polymerase (Roche, Indianapolis, Ind., USA). The amplification products were analyzed by agarose gel electrophoresis, followed by ethidium bromide staining. The marker that was a 100-bp DNA ladder (D-1030; Bioneer, Daejeon, Korea). ELISA: Levels of serum immunoglobulin M (IgM), IgG, and antibodies to C. pneumoniae were also determined by the ELEGANCE TM Chlamydia pneumoniae ELISA test kit, provided by Hitachi Chemical Co., Ltd., Tokyo, Japan. Blank, negative control, positive control, and 100 l of diluted specimen were added to the wells of antigen-fixed plates and allowed to react at 37 C for 60 min. After the samples were washed three times, 100 l each of alkaliphosphatase-labeled anti-human IgM, IgG, and antibodies were added, and the mixture was allowed to react at 37 C for 60 min. After the samples were washed 3 times, 100 l of the substrate solution (p-nitrophenyl phosphate and hydrogen peroxide solution) was added. The mixture was allowed to react at room temperature for exactly 10 min. Then, 25 l of a reactionstopping solution was added, and the p-nitrophenol which was produced was measured at an absorbance of 405 nm. The cut-off absorbance for IgM, IgG, and was set at 0.20, and the index value (ID), which was obtained by dividing the measured absorbance of the specimens and corrected for the expressed value of the positive of the positive control through the cut-off absorbance, was then calculated. Provisionally, an ID of less than 0.9 was considered as negative, and an ID of 0.9 to 1.1 considered borderline (for re-testing); greater than 1.1 was considered to be positive. Subsequently, a more detailed assessment criterion was considered, and this latter criterion was based on the numerical value of the ID (16). MIF antibody: Preparation of EBs: C. pneumoniae antigens were provided by the National Institute of Infectious Diseases (NIID) of Japan. A highly purified EB suspension was prepared (14). MIF assay: The MIF test was performed according to previously described methods (7,17). Slides were prepared with the following antigens: C. trachomatis ATCC VR-571B, C. psittaci Buderigar, and C. pneumoniae TW-183. In this test, C. pneumoniae and C. trachomatis antigens had been treated in order to remove the LPS antigen; however, the LPS antigen was retained in the case of the C. psittaci antigen. Levels of chlamydial IgM, IgG, and antibodies were determined. All IgM reactions were confirmed after the IgG antibodies had been removed with RF Absorbent (Behringwerke, Marburg, Germany). Fourfold titer increases of IgG and/or, and/or a titer of 16 in the case of IgM 295

3 were considered diagnostically significant. RESULTS Culture method and passage: All samples were inoculated in duplicate onto HEp-2 cells grown on coverslips in a shell viral, as described above. The cells were incubated at 35 C in 5% CO 2 for 72 h. On subsequent passages, isolates were identified as C. pneumoniae by staining the cells with a fluorescein-conjugated species-specific antibody. Inclusionnegative cultures were found to be negative; however, the PCR analysis gave positive results, and therefore the culture was passaged again. PCR: The results of the PCR using 53-kDa primers were as follows: 63% PCR positivity of the nasopharyngeal swabs at the acute phase of the outbreak, and up to 94% PCR positivity of the culture samples. However, the presence of the organism was not confirmed by the fluorescein isothiocyanate (FITC) method (Table 1, Fig. 1). Serology test: This ELISA method was utilized to test 19 serum specimens obtained from the patients. The positivity of antibodies for single IgM was 48% (13/27) at the acute phase and 16% (3/19) at the convalescent phase according to the criteria shown in Table 2 (15,16). The rate of ID 3.0 for single-sera IgG was 19% (5/27) at the acute phase and 11% (2/19) at the convalescent phase. The single-serum ID 3.0 accounted for 4% of the samples (1/27) at the acute phase and 5% (1/19) at the convalescent phase. However, when paired sera were considered, no ELISA ID value exceeded 1.35 or 1.0. According to the MIF test, the rate of positive for single IgM was 58% (11/19) at the acute phase and 47% (9/19) at the convalescent phase. The positivity of paired IgG (twofold increase) was 26% (5/19) (Table 3). The positivity ratio for both ELISA and MIF was 37% (7/19) for IgM at the acute phase and 32% (2/19) of for IgM at the convalescent phase (data not shown). COPD patient serology test: Comparison using the antibody titers of COPD patients (n = 42) involved serological testing by using both single and paired serum samples. The COPD patients had been inpatients in the hospital for 3 months, and they exhibited similar symptoms. In ELISA, the single IgM-positive rate among patients was 9% (4/42). The single-serum IgG (ID 3.0) rate was 14% (6/42) and that of (ID 3.0) was 2% (1/42). MIF testing revealed a single IgM (ID 16)-positive rate among patients of 14% (6/42). The single IgG (ID 512) rate was 5% (2/42). The positivity of paired IgG (twofold increase) was 0% (Table 4). The positivity ratio for both the ELISA and MIF at the acute phase was 5% (2/42) for IgM and 2% (1/42) of IgG (data not shown). Comparison of antibody titers: Because there no data were available regarding the healthy population in Korea, we measured the serum of certain members of a healthy sample (n = 37). Normal subjects living in Seoul had been seen in the hospital for common conditions such as headache and backache. Normal subjects on the main island were sampled in a measles immune response study. Table 1. Detection of C. pneumoniae by PCR in the nasopharyngeal swabs of patients No Sex/Age F/13 M/16 F/16 F/16 F/14 F/14 F/14 M/14 F/16 M/14 M/14 F/14 F/16 F/15 F/15 M/16 M/15 M/15 M/16 1st Passage 18th Table 2. Acute C. pneumoniae infection diagnostic criteria Diagnosis Serum MIF ELISA Certain Doubt Single IgM 16 fold ID 1.0 (child) ID 1.6 (adult) IgG 2 fold increased ID 1.35 increased ID 1.00 increased IgG 512 fold ID 3.0 ID 3.0 (ref. 15) Table 3. Analysis of the results of ELISA and MIF methods among patients with respiratory tract infections in the islet Method Serum Specimen Positivity 1st 48% (13/27) IgM 2nd 16% (3/19) 1st 19% (5/27) Single IgG 2nd 11% (2/19) ELISA 1st 4% (1/27) 2nd 5% (1/19) IgG 0% 0% 1st 58% (11/19) Single IgM MIF 2nd 47% (9/19) IgG 26% (5/19) Table 4. Analysis of the results of ELISA and MIF methods among COPD patients Method Serum Diagnosis Positivity IgM Certain 9% (4/42) Single IgG 14% (6/42) Doubt ELISA 2% (1/42) IgG 0% Certain 0% IgM Certain 14% (6/42) Single MIF IgG 5% (2/42) IgG Certain 0% Fig. 1. PCR results of 1st passage with 53-kDa protein primer. M, 100 bp DNA ladder; NC, negative control; PC, positive control. 296

4 Table 5. Summary of antibody tests by ELISA Specimens ELISA positivity (%) IgM IgG Patients in the islet COPD Patients Normal in Seoul Normal in main island The normal control group demonstrated high levels of antibody titers, as compared to those of the islet population (Table 5). However, the antibody titers of the islet sample subsequently rose to those of the normal controls. DISCUSSION Outbreaks of C. pneumoniae infection have been reported in Japan, USA, Denmark, Finland, and Italy (19-22). As reported in Denmark, only 7 out of 593 patients with a TWAR infection were less than 5 years of age (19), whereas most of the patients infected on the Korean islet were between 5 to 17 years of age, which indicated that the outbreak in Korea differed from the European outbreak. Moreover, the age of patients in outbreak in Korea was very close to that in a report by Aldous et al. (21) involving families in the Seattle area. This is the first report of an outbreak of C. pneumoniae on an islet of Korea. The C. pneumoniae-induced antibody response patterns comprise diagnostically useful properties that can be utilized to differentiate a primary infection from a re-infection. Primary C. pneumoniae infection is characterized by an IgM response that appears 2-3 weeks after the first symptoms of the illness. A delayed IgG response appears 6-8 weeks after the illness (10-12). Re-infections are usually recognized by the absence of an IgM response and a more rapidly appearing IgG response than that of a primary infections (3). Continuously detected high levels of IgG antibodies can indicate either chronic or repeated infections, but differentiation between theses two types of infection is often impossible. The PCR positivity of nasopharyngeal swab samples was 63% (12/19) among patients at the acute phase; the PCR positivity increased to 94% (18/19) in the culture samples. However, the presence of the organism was not confirmed by FITC. The ELISA results demonstrated a 48% (13/27) positivity of single IgM antibody at the acute phase of the outbreak; and a 16% (3/19) positivity at the convalescent phase. When the ID value was 3.0, the positivity for single-sera IgG was 19% (5/27), and that for was 4% (1/27) at the acute phase; the respective rates were 11% (2/19) and 5% (1/19) at the convalescent phase. However, in the case of paired sera, no patient demonstrated a 1.35 ELISA ID value of 1.0 either at 2 weeks or 8 weeks after the outbreak. According to the MIF results, the rate of positivity of single IgM was 58% (11/19) at the acute phase, whereas it was 47% (9/19) at the convalescent phase; the corresponding positivity of paired serum IgG was 26% (5/19). Both ELISA and MIF showed rates of positivity of 37% (7/19) for single IgM at the acute phase, and 11% (2/19) at the convalescent phase. Among the MIF-positive results (n = 11), those that were also positive according to ELISA amounted to 64% (7/11) (data not shown). ELISA positivity was determined by the measurement of absorbance; however, which of MIF by subjective determination. Therefore, only slight positivity was still considered as MIF-positive. That was also contained in intermediate range in ELISA, which means the positive rate of ELISA is lower than that of MIF. According to the diagnostic criteria for reinfection provided by the commercial kit, the ID for ELISA would not be expected to be significantly high. The ELISA results were therefore not deemed useful for a diagnosis of C. pneumoniae. Both the PCR and serum reactions gave positive results, but the isolation of C. pneumoniae was not successful. Nevertheless, meaningful results were not obtained in terms of serological testing, when the patients results were compared to those of a healthy sample in Korea. 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