J Infect Chemother xxx (2015) 1e5

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Original article

Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult? Naoyuki Miyashita a, *, Yasuhiro Kawai a, Takaaki Tanaka b, Hiroto Akaike b, Hideto Teranishi b, Tokio Wakabayashi b, Takashi Nakano b, Kazunobu Ouchi b, Niro Okimoto a a b

Department of Internal Medicine I, Kawasaki Medical School, Okayama, Japan Department of Pediatrics, Kawasaki Medical School, Okayama, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2014 Received in revised form 22 January 2015 Accepted 4 March 2015 Available online xxx

The ELNAS Plate Chlamydophila pneumoniae commercial test kit for the detection of anti-C. pneumoniaespecific immunoglobulin M (IgM), IgA and IgG antibodies has become available in Japan recently. To determine the optimum serum collection point for the ELNAS plate in the diagnosis of C. pneumoniae pneumonia, we analyzed the kinetics of the antibody response in patients with laboratory-confirmed C. pneumoniae pneumonia. We enrolled five C. pneumoniae pneumonia cases and collected sera from patients for several months. The kinetics of the IgM and IgG antibody responses were similar among the five patients. Significant increases in IgM and IgG antibody titer between paired sera were observed in all patients. IgM antibodies appeared approximately 2e3 weeks after the onset of illness, reached a peak after 4e5 weeks, and were generally undetectable after 3e5 months. IgG antibodies developed slowly for the first 30 days and reached a plateau approximately 3e4 months after the onset of illness. The kinetics of IgA antibody responses were different among the five patients, and significant increases in IgA antibody titer between paired sera were observed in only two patients. Although the sample size was small, the best serum collection time seemed to be approximately 3e6 weeks after onset of illness when using a single serum sample for the detection of IgM antibodies. Paired sera samples should be obtained at least 4 weeks apart. IgA antibody analysis using ELNAS may not be a useful marker for acute C. pneumoniae pneumonia. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Keywords: Chlamydophila pneumoniae Antibody response ELNAS plate Immunoglobulin M Immunoglobulin G Immunoglobulin A

1. Introduction Chlamydophila pneumoniae, an obligate intracellular human pathogen, has been proven to cause both epidemic and endemic respiratory tract infections worldwide [1e3]. It is a cause of both lower and upper acute respiratory illnesses, and many studies have suggested a possible association of C. pneumoniae infection and acute exacerbations of chronic lung diseases [1e3]. Seroepidemiological studies showing antibody prevalence rates with a range of 50e70% suggest that C. pneumoniae is widely distributed and that nearly everybody is infected with the agent at some time [1,2].

* Corresponding author. Department of Internal Medicine I, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505, Japan. Tel.: þ81 86 225 2111; fax: þ81 86 232 8343. E-mail address: [email protected] (N. Miyashita).

Outbreaks of C. pneumoniae have occurred in closed populations including among families, university students, military trainees, in schools and in nursing homes [1,4]. The most common method still used by investigators for the diagnosis of C. pneumoniae infections is serological testing. In Japan, most clinicians and researchers have access to a widely used commercial serologic test kit, an enzyme-linked immunosorbent assay (ELISA) (Hitazyme C. pneumoniae, Hitachi Chemical Co., Tsukuba, Japan), to detect anti-C. pneumoniae-specific immunoglobulin M (IgM), IgA and IgG antibodies [5,6]. However, several studies evaluating this Hitazyme-ELISA within asymptomatic populations or acute respiratory tract infections in both children and adults demonstrated that positive correlations were not observed between the Hitazyme-ELISA and a microimmunofluorescence (MIF) test, which is the current gold standard for serologic testing of C. pneumoniae infections worldwide [7e13]. The discrepancy was

http://dx.doi.org/10.1016/j.jiac.2015.03.003 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Miyashita N, et al., Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.03.003

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N. Miyashita et al. / J Infect Chemother xxx (2015) 1e5

analyzed using other established serological tests, and it was confirmed that false-positive and false-negative Hitazyme-ELISA reactivity is frequent [8e13]. Thus, most clinicians have difficulty in making a diagnosis of acute C. pneumoniae infection when using the diagnostic criteria of this test. To solve this problem, another commercial test kit for the detection of anti-C. pneumoniae-specific antibodies, ELNAS Plate C. pneumoniae IgM, IgA and IgG (Fujirebio Inc., Tokyo, Japan) has become available recently in Japan [10,12]. The purpose of this study was to determine the optimum serum collection point for the ELNAS for diagnosis of C. pneumoniae pneumonia. We analyzed the kinetics of the antibody response in patients with laboratory-confirmed C. pneumoniae pneumonia.

2. Patients and methods

2.3. MIF test The MIF test was primarily developed for Chlamydia trachomatis by Wang and Grayston, but it was later adapted for use in serodiagnosis of C. pneumoniae infections [15]. The elementary bodies (EBs) of strains TW-183 and KKpn-15 were purified by continuous Urografin (Schering AG, Berlin/Bergkamen, Germany) gradient centrifugation (40e52%). The EBs were resuspended in a solution of 2% yolk sac in phosphate-buffered saline (pH 7.2) containing 0.02% formalin. IgM, IgA and IgG antibodies against C. pneumoniae were detected using commercial fluorescein isothiocyanate-conjugated goat antihuman IgM, IgA and IgG (Medical and Biological Laboratories, Nagoya, Japan). Rheumatoid factor was absorbed with GullSORB (Meridian Bioscience Inc., OH, USA) before IgM titrations. An anti-C. pneumoniae antibody titer of 1:32 for IgM or a fourfold increase in IgA or IgG was considered to indicate acute infection.

2.1. Study population 2.4. Culture This study was conducted as part of our wide-ranging studies on pneumonia, which are investigating the clinical features and evaluating the usefulness of diagnostic methods for the diagnosis of atypical pneumonia. All adolescent and adult patients with community-acquired pneumonia (CAP) who visited Kawasaki Medical School Hospital or Kawasaki Medical School Kawasaki Hospital, Okayama, Japan, from April 2010 to November 2014 were enrolled in this study. The diagnosis was based on clinical signs and symptoms (cough, fever, productive sputum, dyspnea, or chest pain) and radiographic pulmonary abnormalities that were at least segmental and not caused by pre-existing or other known causes. Exclusion criteria included immunosuppressive illness (i.e., HIV positive, neutropenia secondary to chemotherapy, use of >20 mg/ day prednisone or other immunosuppressive agents, and history of organ transplant); hospitalization in the preceding 30 days; residence in a nursing home or extended care facility; and active tuberculosis. During the study period, 478 CAP cases were assessed using the microbiological tests. Paired serum samples were collected at intervals of at least 4 weeks after onset. Five patients were diagnosed with C. pneumoniae pneumonia by MIF and ELNAS tests. We followed up and collected serum samples from these five patients for several months. Informed consent was obtained from all patients, and the study protocol was approved by the Ethics Committee at Kawasaki Medical School (approval number 679).

2.2. ELNAS plate C. pneumoniae The ELNAS Plate C. pneumoniae for IgM, IgA and/or IgG antibodies is an indirect solid-phase enzyme immunoassay (EIA) based on an antigen from C. pneumoniae devoid of lipopolysaccharide (LPS). The results for IgA and IgG are expressed as enzyme immunounits (EIU), which are calculated as follows: (Asample  Ablank)/ (Acalibrator  Ablank)  n. The assays are calibrated to correspond to the inverted titers of the MIF test, with n ¼ 130 for the IgG EIA and n ¼ 30 for the IgA EIA. A level of 30 EIU for IgG is considered the limit of detection (corresponding to a titer of 32 in the MIF test) and a level of 8 EIU in the IgA EIA is the corresponding limit of detection (titer of 8 in the MIF test) [14]. The criterion for a diagnostically significant change in EIU values for IgG and IgA is a 1.5-fold change in EIU in the zones below 130 EIU for IgG and 50 EIU for IgA [14]. When the first sample shows an EIU value of 130 or more for IgG and 50 for IgA, a 1.3-fold change is considered significant [14]. The results of C. pneumoniae IgM EIA are expressed as a signal/cutoff (S/CO) ratio after subtraction of the blank value. Samples with an S/CO ratio of more than 1.1 are considered positive.

Nasopharyngeal swab specimens for culture were placed in a sucrose-phosphate-glutamate transport medium. Each swab specimen was sonicated and briefly centrifuged (900  g for 10 min), and the supernatant was overlayed on confluent monolayers of HEp-2 cells grown on round coverslips (14 mm in diameter) set in a 24-well plastic cell culture plate. The plate was centrifuged at 1200  g for 60 min at room temperature. Next, 1 mL of culture medium consisting of Eagle's minimal essential medium (Nissui Pharmaceuticals Co, Tokyo, Japan), 10% heat-inactivated fetal calf serum (Gibco BRL Life Technologies Inc, Grand Island, NY, USA), and cycloheximide (Nacalai Tesque Inc, Tokyo, Japan) at a final concentration of 1 mg/mL was applied. Then, the plates were incubated in 5% CO2 at 35  C for 72 h, and all specimens were passaged twice. Following incubation, a genus-specific fluorescein isothiocyanate-conjugated monoclonal antibody (Chlamydia FA Seiken; Denka Seiken, Tokyo, Japan) and C. pneumoniae speciesspecific monoclonal antibodies were used to stain inclusions. Inclusions were observed using a Nikon epifluorescence microscope (Nikon, Tokyo, Japan) at 200 or 400 magnification. 2.5. Real-time polymerase chain reaction assay Real-time polymerase chain reaction (PCR) was performed using an ABI Prism 7700 sequence detector system (Applied Biosystems) as reported by Apfalter et al. [16]. The ompA gene was used as the target for amplification. The PCR product generated was 85 base pairs in length, and the sequences of the primer and the TaqMan probe were designed as described by Apfalter et al. [16]. PCR was performed in 96 well MicroAmp optical plates (Applied Biosystems). The reaction mixtures consisted of 25 mL of TaqMan Universal Master mix including dUTP and N-glycosylase (AmpErase UNG; Applied Biosystems), each of the primers at a concentration of 300 nM, and 200 nM TaqMan probe in a total reaction volume of 50 mL for the clinical samples. Briefly, the PCR conditions were as follows: 2 min at 50  C and 10 min at 95  C, followed by 40 cycles at 95  C for 15 s and at 60  C for 1 min. Sequence Detection Systems 1.6.3 was used for analysis after real-time PCR. A positive result was accepted if three tests were positive. If a positive reaction could not be repeated, the sample was judged to be negative. 3. Results 3.1. Patient characteristics The characteristics of the five patients with C. pneumoniae pneumonia are shown in Table 1. All cases were IgM positive and

Please cite this article in press as: Miyashita N, et al., Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.03.003

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Table 1 Clinical characteristics of five patients with laboratory-confirmed Chlamydophila pneumoniae pneumonia on first visit to our hospital. Characteristics

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Age, years Gender Co-morbid illness PSI risk classes A-DROP score JRS differential diagnosis score Laboratory data WBC/mm3 C-reactive protein, mg/dL Culture for C. pneumonia PCR for C. pneumonia Serum collection time after onset of illness, days First-choice antibiotic Effective antibiotic

19 Female Asthma I 0 5

30 Female None I 0 6

36 Female None I 0 5

40 Male None I 0 6

42 Female None I 0 6

5480 0.91 Negative Negative 8, 26, 62, 120 Macrolide Macrolide

7470 5.21 Negative Negative 11, 22, 35, 66, 101, 132 Minocycline Minocycline

7180 4.82 Negative Negative 16, 23, 30, 36, 76, 124, 166 Minocycline Minocycline

6620 5.31 Negative Negative 14, 24, 32, 55, 68, 110, 144, 186 Fluoroquinolone Fluoroquinolone

6410 0.77 Negative Negative 10, 19, 45, 79, 114, 142, 181 Macrolide Macrolide

JRS ¼ Japan Respiratory Society, PCR ¼ polymerase chain reaction, PSI ¼ pneumonia severity index.

significant IgG antibody seroconversion (fourfold increase in MIF test and 1.5-fold change in ELNAS). Culture and PCR for C. pneumoniae were negative in all cases. No mixed infection with other pathogens was observed. The severity of pneumonia was very mild in all cases assessed by the use of a clinical severity scale, the pneumonia severity index (PSI) published by the Infectious Diseases Society of America (IDSA) [17] and the A-DROP system proposed by the Japan Respiratory Society (JRS) [18]. All cases were judged as atypical pneumonia by scoring systems for differentiating bacterial pneumonia and atypical pneumonia in the JRS guidelines [18]. All cases showed air-space pneumonia with segmental homogenous consolidation. Chest physicians could not detect any abnormal shadows in chest radiographs in two cases, but chest computed tomography showed homogenous consolidation in these patients (Fig. 1). One patient received fluoroquinolone, two patients received macrolides and two patients received minocycline. All C. pneumoniae pneumonia infections were cured and no relapse was observed. 3.2. Antibody responses Positive and negative correlations were observed between the ELNAS and a MIF test. The kinetics of IgM and IgG antibody responses were similar among the five patients. Significant increases in IgM and IgG antibody titer between paired sera were observed in

Fig. 2. IgM antibody responses in five patients with laboratory-confirmed Chlamydophila pneumoniae pneumonia.

all patients. IgM antibodies appeared approximately 2e3 weeks after the onset of illness, reached a peak after 4e5 weeks and were generally undetectable after 3e5 months (Fig. 2). IgG antibodies developed slowly for the first 30 days and reached a plateau at approximately 3e4 months after the onset of illness (Fig. 3). The kinetics of IgA antibody responses were different among the five patients, and significant increases in IgA antibody titer between paired sera were observed in only two patients (Fig. 4). IgA

Fig. 1. Patient number 5 (42-year-old female). Chest radiograph showing no abnormal shadows. Chest computed tomography showing a homogeneous infiltrate in the left segment 10 area.

Please cite this article in press as: Miyashita N, et al., Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.03.003

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Fig. 3. IgG antibody responses in five patients with laboratory-confirmed Chlamydophila pneumoniae pneumonia.

antibody titer was positive from the onset of illness in two patients, but IgG antibody titer was negative in both patients. Thus, these cases seems to be non-specific reaction of IgA antibody.

4. Discussion Regarding serological tests for C. pneumoniae infections, only the MIF test is strongly recommended [19]. However, most Japanese clinicians and researchers do not use the MIF test for routine diagnosis because this assay is technically demanding, timeconsuming, and difficult for antigen preparation and interpretation. Since 2014, a partially automated EIA kit, ELNAS Plate C. pneumoniae, has been available for the detection of antiC. pneumoniae-specific IgM, IgA and IgG antibodies, and specific diagnosis of C. pneumoniae infection was anticipated. In this study, we focused on investigating the antibody responses in patients with C. pneumoniae pneumonia by ELNAS. The present study together with our previous studies demonstrated that the results from the ELNAS correlated well with those of an MIF test [10e13]. The same results were also demonstrated by Persson and Boman with the specificity of both the ELNAS and MIF tests at 99% [20]. Among the Ig classes, IgM antibodies are helpful for the rapid diagnosis of acute C. pneumoniae infections because of the early serologic response to C. pneumoniae compared with IgA or IgG antibodies [1,19]. In a clinical setting, a single rather than acute and convalescent samples is available for the diagnosis of acute infections. Our results, together with previous studies [15,19], demonstrated that the best serum collection time seemed to be approximately 3e6 weeks after the onset of illness when using a

Fig. 4. IgA antibody responses in five patients with laboratory-confirmed Chlamydophila pneumoniae pneumonia.

single serum sample for the detection of IgM antibodies. Our results demonstrated that paired sera samples should be obtained at least 4 weeks apart, as recommended by the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada) [19]. The usefulness of IgA antibodies as a diagnostic marker in acute or chronic C. pneumoniae infections has not been demonstrated [15,19]. Our results supported these results even though we used a different serological test; therefore, for IgA antibodies, the ELNAS may not be a useful marker for acute C. pneumoniae pneumonia. To avoid confusion of interpretation with IgA results, physicians probably should not measure the IgA antibody. The symptoms and infiltration shadows on chest radiograph were disappeared within 2 weeks after initiation of appropriate antibiotics in all patients and IgM antibody titers reached a peak after resolution of pneumonia. The IgM antibody titer was higher in patients with wide-range infiltrate than in patients with narrowrange infiltrate. C. pneumoniae infection does not induce good protective immunity, and re-infection may occur. In cases with primary infection, IgM antibody appear 2e3 weeks after the onset of illness and IgG antibody appear 6e8 weeks after the onset of illness, whereas in cases of re-infection, IgM antibody may be absent, or of low titer if present, and IgG antibody appear earlier, within 1e2 weeks after the onset of illness [1e3,19]. According to these criteria, our five cases were classified as primary infection. However, these kinetics of the antibody response were determined by MIF test. Thus, criteria of the primary infection and re-infection for the ELNAS is needed. Among the diagnostic methods for C. pneumoniae infections, culturing, antigen detection assays and molecular techniques are still not routine because these tests require specialized laboratories and/or are expensive, time-consuming and labor-intensive. Thus, serological analysis represents the current routine method for the diagnosis of C. pneumoniae infections. In addition, several studies together with our study confirmed the lack of correlation of serological methods with culture and/or PCR assays observed in this study [21e26]. Why is the diagnosis of C. pneumoniae pneumonia difficult? There are several reasons. First, appearance of anti-C. pneumoniae IgM antibodies is approximately 2e3 weeks after the onset of illness, and positive-IgM results may be detected after the resolution of pneumonia. The IgG antibody response might be missed if convalescent sera were obtained too soon (i.e., earlier than 3 weeks after the onset of illness) because it takes 4e8 weeks for a significant increase in IgG antibodies. Second, most clinicians and researchers are used to using the Hitazyme-ELISA kit for the diagnosis of C. pneumoniae infection in Japan. Hermann and coworkers evaluated 11 commercial serological tests, including the HitazymeELISA kit (Elegance), for IgG antibodies in a healthy population [7]. The sensitivities and specificities of the MIF tests were high, but a poor correlation was found for the results of the Hitazyme-ELISA in comparison with those of the MIF test and other serological tests [7]. In addition, it is well known that Hitazyme-ELISA IgM antibody frequently shows false-positive findings in both healthy subjects and patients with respiratory tract infections [8e13]. Third, our studies demonstrated that C. pneumoniae is a low-frequency etiologic pathogen in both hospitalized community-acquired pneumonia and hospitalized nursing and healthcare-associated pneumonia. The severity of C. pneumoniae pneumonia is very mild [1,27]. Asymptomatic or mildly symptomatic C. pneumoniae infections are common [1,27], and pneumonia shadows may be quite difficult to detecting from routine chest radiographs, as shown in this study. Thus, many C. pneumoniae pneumonia cases may be missed.

Please cite this article in press as: Miyashita N, et al., Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.03.003

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A limitation of our study was its small sample size owing to the difficulty in enrolling patients with C. pneumoniae pneumonia who could be followed-up for several months. Further prospective studies are needed to clarify the antibody responses of ELNAS in patients with C. pneumoniae pneumonia. In conclusion, our results demonstrated that the optimum serum collection time for the diagnosis of C. pneumoniae pneumonia seemed to be approximately 3e6 weeks after the onset of illness when using a single serum sample for the detection of IgM antibodies. Using paired sera samples for the detection of IgM and IgG antibodies, samples should be collected at least 4 weeks apart. Conflict of interest The authors have no conflicting interests. Acknowledgments This work was supported by MEXT KAKENHI (19591190 and 21591304) and Project Research Grants from Kawasaki Medical School (13-401, 14-402, 15-405A, 16-405M, 17-402M, 18-401, 19402M, 20-4030). References [1] Kuo CC, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 1995;8:451e61. [2] Blasi F, Tarsia P, Aliberti S. Chlamydophila pneumoniae. Clin Microbiol Infect 2009;15:29e35. [3] Burillo A, Bouza E. Chlamydophila pneumoniae. Infect Dis Clin North Am 2010;24:61e71. [4] Conklin L, Adjemian J, Loo J. Investigation of a Chlamydia pneumoniae outbreak in a federal correctional facility in Texas. Clin Infect Dis 2013;57:639e47. [5] Kishimoto T, Matsushima T, Morikawa T, Kawagoe K. Assay of specific antiChlamydia pneumoniae antibodies by ELISA method. 3. Setting of serological criteria. Kansenshogaku Zasshi 1999;73:457e66 (in Japanese). [6] Kishimoto T, Ando S, Numazaki K, Ouchi K, Yamazaki T, Nakahama C. Assay of Chlamydia pneumoniae- specific IgM antibodies by ELISA method e reduction of non-specific reaction and resetting of serological criteria by measuring IgM antibodies-. Jpn J Infect Dis 2009;62:260e4. [7] Hermann C, Graf K, Groh A, Straube E, Hartung T. Comparison of eleven commercial tests for Chlamydia pneumoniae-specific immunoglobulin G in asymptomatic healthy individuals. J Clin Microbiol 2002;40:1603e9. [8] Miyashita N, Obase Y, Fukuda M, Shouji H, Mouri K, Yagi S, et al. Evaluation of serological tests detecting Chlamydophila pneumoniae-specific immunoglobulin M antibody. Intern Med 2006;45:1127e31. [9] Miyashita N, Ouchi K, Kawasaki K, Komura H, Kawai Y, Obase Y, et al. Evaluation of enzyme-linked immunosorbent assay for Chlamydophila pneumoniae-specific immunoglobulin M in acute respiratory tract infection. Respirology 2008;13:299e302.

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Please cite this article in press as: Miyashita N, et al., Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.03.003

Antibody responses of Chlamydophila pneumoniae pneumonia: Why is the diagnosis of C. pneumoniae pneumonia difficult?

The ELNAS Plate Chlamydophila pneumoniae commercial test kit for the detection of anti-C. pneumoniae-specific immunoglobulin M (IgM), IgA and IgG anti...
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