J Infect Chemother xxx (2015) 1e3
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Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
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Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR 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 1, 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 18 November 2014 Received in revised form 30 January 2015 Accepted 9 February 2015 Available online xxx
A rapid antigen kit for the detection of the Mycoplasma pneumoniae ribosomal protein L7/L12 using an immunochromatographic assay, Ribotest Mycoplasma, became available in Japan in 2013. To determine the sensitivity of Ribotest compared with real-time polymerase chain reaction (PCR), we prospectively performed these two tests simultaneously in adolescent and adult patients with community-acquired pneumonia (CAP). In addition, we retrospectively analyzed the theoretical sensitivity of Ribotest using M. pneumoniae PCR-positive specimens from previous studies. In prospective study, 118 CAP cases were enrolled, and 16 cases were diagnosed as M. pneumoniae pneumonia; eight cases were PCR-positive, one case was culture positive, and all cases demonstrated a four-fold increase in antibody titer. Ribotest was positive in 15 cases; five cases were PCR positive and 10 cases were PCR negative. For the PCR was control test, the sensitivity, specificity, and overall agreement with Ribotest were 62.5%, 90.9%, and 88.9%, respectively. In the retrospective study, we used 1110 M. pneumoniae PCR-positive specimens, which are collected from pediatric patients with respiratory tract infection who visited 65 institutions throughout Japan. Using a cut-off level for the Ribotest of 8.3 104 copy/mL in transport medium, 667 (60.0%) specimens were theoretically positive. In conclusion, our prospective and retrospective results demonstrated that the diagnostic sensitivity of Ribotest compared with PCR was not high, at approximately 60%. Thus, treatment decisions about M. pneumoniae pneumonia should be based on clinical findings such as Japanese Respiratory Society scoring system and not on Ribotest results alone. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycoplasma pneumoniae Community-acquired pneumonia Ribotest Mycoplasma Real-time polymerase chain reaction Sensitivity
Mycoplasma pneumoniae is a major cause of respiratory tract infections (RTIs) in school-age children and young adults [1e5] and is the second leading pathogen of community-acquired pneumonia (CAP) after Streptococcus pneumoniae [3,4]. Pneumonia due to M. pneumoniae is usually mild, but 3e4% of those infected with M. pneumoniae are known to develop severe, life-threatening pneumonia with respiratory failure [6,7]. A specific diagnosis of M. pneumoniae infection is important because treatment with routinely used b-lactam antibiotics is ineffective against such organisms that lack a cell wall.
* Corresponding author. Department of Internal Medicine 1, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama City, Okayama 700-8505, Japan. Tel.: þ81 86 225 2111; fax: þ81 86 232 8343. E-mail address: [email protected] (N. Miyashita).
A rapid antigen kit for the detection of M. pneumoniae L7/L12 ribosomal protein using an immunochromatographic assay, Ribotest Mycoplasma (Asahi Kasei Pharma Co., Tokyo, Japan), became available in Japan in August 2013 [8e10]. L7/L12 ribosomal protein is a component of the 50S ribosome that is abundant in all bacteria and is specific for each bacterial species [11]. It interacts with translation factors during protein biosynthesis in bacteria. However, available data on the sensitivity of this kit are limited with only clinical performance trials used in the application for approval [10]. The main purpose of the present study was to determine the sensitivity of Ribotest compared with real-time polymerase chain reaction (PCR) as the gold standard. We prospectively performed these two tests simultaneously in adolescent and adult patients with CAP. In addition, we retrospectively analyzed theoretical sensitivity using M. pneumoniae PCR-positive specimens from previous studies.
http://dx.doi.org/10.1016/j.jiac.2015.02.007 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., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
2
N. Miyashita et al. / J Infect Chemother xxx (2015) 1e3
The prospective study was conducted by Kawasaki Medical School Hospital and Kawasaki Medical School Kawasaki Hospital from November 2013 to October 2014. We enrolled adolescent and adult patients with CAP. 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. Informed consent was obtained from all patients, and the study protocol was approved by the Ethics Committee of Kawasaki Medical School (approval number 1279). In the retrospective study, we used real-time PCR-positive specimens that were collected from pediatric patients with RTIs who visited 65 institutions throughout Japan, participating in the Atypical Pathogen Study Group from January 2008 to December 2013 [12,13]. A complete list of participating facilities is provided in the Appendix. Cultivation of M. pneumoniae was carried out with pleuropneumonia-like organism broth (PPLO; Oxoid, Franklin, NJ, USA) supplemented with 0.5% glucose (Wako Pure Chemicals Inc., Osaka, Japan), 20% mycoplasma supplement-G (Oxoid), and 0.0025% phenol red (Sigma-Aldrich Co. LLC., St. Louis, MO, USA) using nasopharyngeal swab specimens [14]. DNA then was extracted by using a QIAamp DNA Mini Kit (QIAGEN K. K., Tokyo, Japan) in accordance with the manufacturer's instructions. M. pneumonia DNA was detected by real-time PCR targeting a conserved part of the gene encoding the P1 adhesin gene [14]. A search for mutations at sites 2063, 2064, and 2617 in the M. pneumoniae 23S rRNA domain V gene region was performed using a direct sequencing method in isolates or samples with a positive PCR result, as reported previously [14]. Ribotest Mycoplasma was performed in accordance with the manufacturer's instructions. Antibodies to M. pneumoniae were measured using a particle agglutination test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan). During the prospective study period, 118 CAP cases were assessed using the microbiological tests. Sixteen cases were diagnosed as M. pneumoniae pneumonia; eight cases were PCR-positive, one case was culture-positive, and all cases demonstrated a fourfold increase in antibody titer. One culture-positive specimen was also PCR-positive. During the retrospective study period, 1110 cases were M. pneumoniae PCR-positive. Categorical variables of age, gender, and mutation type of the eight patients in the prospective study and the 1110 patients in retrospective study with PCR-positive M. pneumoniae infection are presented in Table 1. Among the PCR-positive cases, five cases in prospective study and 801 cases in retrospective study were found to be infected with macrolide-resistant M. pneumoniae. The correlations between Ribotest and real-time PCR results using 118 CAP cases are presented in Table 2. Ribotest was positive in 15 cases; five cases were PCR positive and 10 cases were PCR negative. When the PCR was used as the control test, the sensitivity, specificity, and overall agreement of Ribotest were 62.5%, 90.9%, and 88.9%, respectively. Among the 15 Ribotest-positive cases, six cases were serology positive and nine cases were serology negative. When the serology was used as the control test, the sensitivity, specificity, and overall agreement of Ribotest were 37.5%, 91.1%, and 83.8%, respectively. One serology positive case was observed in 10 PCR-negative and Ribotest-positive cases. Because the M. pneumoniae PCR-positive sample size in the prospective study was too small to evaluate the sensitivity of
Table 1 Categorical variables of age, gender, and mutation type of patients with PCR-positive M. pneumoniae infection in prospective and retrospective studies. Variables
Prospective study
No. of patients 8 Mean age (range), years 19.8 (16e24) No. of males/females 4/4 No. of resistant cases 5 No. of point mutation in domain V of 23S rRNA A2063Ga 5 A2063T 0 A2064G 0 A2064C 0 C2617G 0 a
Retrospective study 1110 7.6 (0e15) 544/566 801 772 20 5 3 1
A-to-G transition at position 2063 in domain V on the 23S rRNA gene.
Ribotest, we conducted the retrospective analysis using PCRpositive specimens from previous studies [12,13]. The cut-off level of Ribotest has reported as 250 copy/mL. We evaluated the detection limit of Ribotest independently in our laboratory and confirmed the same cut-off level. The swab specimens collected from patients were suspended in 3.0 mL transport medium, and then DNA was extracted using 300 mL medium. The extracted DNA was eluted in a total volume of 100 mL, and then 5 mL was used for PCR assay. Thus, the cut-off level of Ribotest was calculated as 8.3 104 copy/mL in transport medium and 2.5 105 copy/swab, respectively. Using this cut-off level, the theoretical positive number of Ribotest was calculated. Among 1110 M. pneumoniae PCR-positive specimens, 667 (60.0%) specimens were theoretically positive. In a clinical performance trial with 33 M. pneumoniae PCRpositive specimens out of 176 CAP cases, it was demonstrated that the sensitivity, specificity, and overall agreement of Ribotest compared with a PCR assay were 57.6%, 91.6%, and 85.2%, respectively [10]. These data are consistent with our present results despite the small sample size. In addition, the theoretical sensitivity with 60% of our retrospective study was also similar to the sensitivity of clinical performance trial. CAP remains a significant cause of morbidity and death worldwide. Rapid diagnostic tests are useful tools in the early presumptive diagnosis of CAP caused by certain pathogens. These results can be used to initiate appropriate antibiotic treatment and prevent antimicrobial resistance through the use of narrowspectrum antibiotics. Among the microbiologic tests, serological testing is the most common tool for the diagnosis of M. pneumoniae infection. However, this test requires paired serum samples with a 2e4 week interval and provides only a retrospective diagnosis. A rapid and simple serologic test, ImmunoCard Mycoplasma (Meridian Bioscience, Cincinnati, OH, USA), was developed for the detection of IgM antibodies [15], and this kit has become widely used in Japan. However, our study indicated that the ImmunoCard test suffers from several shortcomings. First, this kit showed many false-positive reactions in healthy adults. Second, IgM antibodies persist for several weeks and months. These findings suggest it is not best practice to base a diagnosis of M. pneumoniae pneumonia on a single assay for IgM alone [16]. Table 2 Correlations between Ribotest and real-time PCR results using 118 CAP cases in the prospective study. Real-time PCR
Ribotest Mycoplasma
Total
Positive
Negative
Positive Negative
5 3
10 100
15 103
Total
8
110
118
Please cite this article in press as: Miyashita N, et al., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
N. Miyashita et al. / J Infect Chemother xxx (2015) 1e3
In this situation, another rapid and simple kit, Ribotest Mycoplasma, became available for the diagnosis of M. pneumoniae infection. Our prospective and retrospective studies and the previous study [10] demonstrated that the diagnostic sensitivity of Ribotest was not high compared with PCR, at approximately 60%. Physicians using Ribotest should have a clear understanding of its advantages and disadvantages. It is expected that the approval of improved version of Ribotest, second generation and third generation tests. The limitation of our study was its small sample size for PCRpositive specimens. Further prospective studies are needed to clarify the sensitivity and specificity of Ribotest in patients with CAP. The results from the retrospective analysis are just supplementary support. In addition, we could not evaluate the factors affecting the results of Ribotest. In conclusion, our prospective and retrospective results demonstrated that the diagnostic sensitivity of Ribotest compared with PCR was approximately 60%. Thus, treatment decisions about M. pneumoniae pneumonia should be based on clinical findings such as Japanese Respiratory Society scoring system [4,5] and not on Ribotest results alone. Conflict of interest None. Acknowledgments This study was supported in part 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, 19-402M, 20-4030). Appendix. Participating facilities The Atypical Pathogen Study Group is formed by Hideki Asaki (Asaki Pediatric Clinic); Kazutoyo Asada (National Mie Hospital); Tomohiro Ichimaru (Saga Prefectural Hospital Koseikan); Toshio Ineda (Inada Clinic); Takuya Inoue (Chayamati Pediatric Clinic); Masakazu Umemoto (Umemoto Pediatric Clinic); Kanetsu Okura (Okura Clinic); Kenji Okada (Fukuoka National Hospital); Takashige Okada (Okada Pediatric Clinic); Teruo Okafuji (Okafuji Pediatric Clinic); Yasuko Okamoto (Okamoto Clinic); Shinichiro Oki (Higashisaga National Hospital); Keiko Oda (Kawasaki Medical School Kawasaki Hospital); Jin Ochiai (Ochiai Pediatric Clinic); Seiko Obuchi (Obuchi Clinic); Yoji Kanehara (Kanehara Pediatric Clinic); You Kanematsu (Kanematsu Pediatric Clinic); Shoji Kouno (Shomonoseki City Central Hospital); Makoto Kuramitsu (Aoba Pediatric Clinic); Katsuji Kuwakado (Kurashiki Central Hospital); Satoshi Kuwano (Kuwano Kids Clinic); Tatuso Koga (Koga Pediatric Clinic); Hayashi Komura (Komura Pediatric Clinic); Hiroshi Sakata (Asahikawa-Kosei General Hospital); Takahisa Sakuma (Sakuma Pediatric Clinic); Kazuhide Shiotsuki (Shiotsuki Internal Medicine Pediatric Clinic); Yasushi Shimada (Shimada Clinic); Makio Sugita (Kurashiki Riverside Hospital); Toru Sugimura (Sugimura Pediatric Clinic); Shumei Takeda (Takeda Pediatric Clinic); Isao Tanaka (Mizushima Central Hospital); Hiroyuki Tanaka (Tanaka Family Clinic); Naohumi Tomita (Tomita Clinic); Kensuke Nagai (Nagai Pediatric Internal Medicine Clinic); Yoshikuni Nagao (Mabi Memorial Hospital); Hidekazu Nakashima (Kojima Central Hospital); Tadashi Nagata (Nagata Pediatric Clinic); Kimiko Nakamura (Enoura Clinic);
3
Kazuyo Nomura (Kama Red Cross Hospital); Kanoko Hashino (Hashino Pediatric Clinic); Yuko Hirata (Hirata Internal Medicine Pediatric Clinic); Kazumi Hiraba (Mokubo Pediatric Clinic); Takuji Fujisawa (Fujisawa Pediatric Clinic); Akiko Maki (Hashima Pediatric Clinic); Toshinobu Matsuura (Yoshino Pediatric Clinic); Nobuyoshi Mimaki (Kurashiki Medical Center); Tatsuhiko Moriguchi (Sakai Hospital Kinki University Faculty of Medicine); Shigeru Mori (Momotaro Clinic); Yoichiro Yamaguchi (Yamaguchi Pediatric Clinic); Syuji Yamada (Yamada Pediatric Clinic); Teruyo Fujimi (Fujimi Clinic); Norio Tominaga (Isahaya Health Insurance General Hospital); Syunji Hasegawa (Yamaguchi University Graduate School of Medicine); Kiyoko Nishimura (Nishimura Pediatric Clinic); Mihoko Mizuno (Daido Clinic); Jiro Iwamoto (Iizuka Hospital); Toshiyuki Iizuka (Hakuai Hospital); Shigeru Yamamoto (Daido Municipal Pediatric Clinic); Tomomichi Kurasaki (Kurosaki Pediatric Clinic); Tadashi Matsubayashi (Seirei Hamamatsu General Hospital).
References [1] Waites KB, Talkington DF. Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 2004;17:697e728. [2] Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev 2008;32:956e73. [3] Miyashita N, Fukano H, Mouri K, Fukuda M, Yoshida K, Kobashi Y, et al. Community-acquired pneumonia in Japan: a prospective ambulatory and hospitalized patient study. J Med Microbiol 2005;54:395e400. [4] The committee for the Japanese Respiratory Society guidelines in management of respiratory infections. The Japanese Respiratory Society guideline for the management of community-acquired pneumonia in adults. Respirology 2006;11(Suppl. 3):79e133. [5] Miyashita N, Kawai Y, Akaike H, Ouchi K, Hayashi T, Kurihara T, et al. Influence of age in the clinical differentiation of atypical pneumonia in adults. Respirology 2012;17:1073e9. [6] Izumikawa K, Izumikawa K, Takazono T, Kosai K, Morinaga Y, Nakamura S, et al. Clinical features, risk factors and treatment of fulminant Mycoplasma pneumoniae pneumonia: a review of the Japanese literature. J Infect Chemother 2014;20:181e5. [7] Miyashita N, Obase Y, Ouchi K, Kawasaki K, Kawai Y, Kobashi Y, et al. Clinical features of severe Mycoplasma pneumoniae pneumonia in adults admitted to an intensive care unit. J Med Microbiol 2007;56:1625e9. [8] Lucer CA, Wong KP. Conformational stability of ribosomal protein L7/L12: effects of pH, temperature, and guanidinium chloride. Biochemistry 1980;19: 176e83. [9] Kolberg J, Hoiby EA, Lopez R, Sletten K. Monoclonal antibodies against Streptococcus pneumoniae detect epitopes on eubacterial ribosomal proteins L7/L12 and on streptococcal elongation factor Ts. Microbiology 1997;143: 55e61. [10] Hamaguchi Y, Odawara F. Novel antigen kit by detection of the ribosomal proteins L7/L12. Mycoplasma antigen lit; Ribotest Mycoplasma. Med Sci Dig 2014;40:506e10 [in Japanese]. [11] Gudkov AT. The L7/L12 ribosomal domain of the ribosome: structural and functional studies. FEBS Lett 1997;407:253e6. [12] Kawai Y, Miyashita N, Kubo M, Akaike H, Kato A, Nishizawa Y, et al. Therapeutic efficacy of macrolides, minocycline, and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae pneumonia in pediatric patients. Antimicrob Agents Chemother 2013;57:2252e8. [13] Kawai Y, Miyashita N, Kubo M, Akaike H, Kato A, Nishizawa Y, et al. Nationwide surveillance of macrolide-resistant Mycoplasma pneumoniae infection in pediatric patients. Antimicrob Agents Chemother 2013;57:4046e9. [14] Miyashita N, Akaike H, Teranishi H, Ouchi K, Okimoto N. Macrolide-resistant Mycoplasma pneumoniae pneumonia in adolescents and adults: clinical findings, drug susceptibility and therapeutic efficacy. Antimicrob Agents Chemother 2013;57:5181e5. [15] Alexander TS, Gray LD, Kraft JA, Leland DS, Nikaido MT, Willis DH. Performance of Meridian ImmunoCard Mycoplasma test in a multicenter clinical trial. J Clin Microbiol 1996;34:1180e3. [16] Miyashita N, Kawai Y, Yamaguchi T, Ouchi K, Oka M, Atypical Pathogen Study Group. Clinical potential of diagnostic methods for the rapid diagnosis of Mycoplasma pneumoniae pneumonia in adults. Eur J Clin Microbiol Infect Dis 2011;30:439e46.
Please cite this article in press as: Miyashita N, et al., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Note
Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR 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 1, 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 18 November 2014 Received in revised form 30 January 2015 Accepted 9 February 2015 Available online xxx
A rapid antigen kit for the detection of the Mycoplasma pneumoniae ribosomal protein L7/L12 using an immunochromatographic assay, Ribotest Mycoplasma, became available in Japan in 2013. To determine the sensitivity of Ribotest compared with real-time polymerase chain reaction (PCR), we prospectively performed these two tests simultaneously in adolescent and adult patients with community-acquired pneumonia (CAP). In addition, we retrospectively analyzed the theoretical sensitivity of Ribotest using M. pneumoniae PCR-positive specimens from previous studies. In prospective study, 118 CAP cases were enrolled, and 16 cases were diagnosed as M. pneumoniae pneumonia; eight cases were PCR-positive, one case was culture positive, and all cases demonstrated a four-fold increase in antibody titer. Ribotest was positive in 15 cases; five cases were PCR positive and 10 cases were PCR negative. For the PCR was control test, the sensitivity, specificity, and overall agreement with Ribotest were 62.5%, 90.9%, and 88.9%, respectively. In the retrospective study, we used 1110 M. pneumoniae PCR-positive specimens, which are collected from pediatric patients with respiratory tract infection who visited 65 institutions throughout Japan. Using a cut-off level for the Ribotest of 8.3 104 copy/mL in transport medium, 667 (60.0%) specimens were theoretically positive. In conclusion, our prospective and retrospective results demonstrated that the diagnostic sensitivity of Ribotest compared with PCR was not high, at approximately 60%. Thus, treatment decisions about M. pneumoniae pneumonia should be based on clinical findings such as Japanese Respiratory Society scoring system and not on Ribotest results alone. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycoplasma pneumoniae Community-acquired pneumonia Ribotest Mycoplasma Real-time polymerase chain reaction Sensitivity
Mycoplasma pneumoniae is a major cause of respiratory tract infections (RTIs) in school-age children and young adults [1e5] and is the second leading pathogen of community-acquired pneumonia (CAP) after Streptococcus pneumoniae [3,4]. Pneumonia due to M. pneumoniae is usually mild, but 3e4% of those infected with M. pneumoniae are known to develop severe, life-threatening pneumonia with respiratory failure [6,7]. A specific diagnosis of M. pneumoniae infection is important because treatment with routinely used b-lactam antibiotics is ineffective against such organisms that lack a cell wall.
* Corresponding author. Department of Internal Medicine 1, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama City, Okayama 700-8505, Japan. Tel.: þ81 86 225 2111; fax: þ81 86 232 8343. E-mail address: [email protected] (N. Miyashita).
A rapid antigen kit for the detection of M. pneumoniae L7/L12 ribosomal protein using an immunochromatographic assay, Ribotest Mycoplasma (Asahi Kasei Pharma Co., Tokyo, Japan), became available in Japan in August 2013 [8e10]. L7/L12 ribosomal protein is a component of the 50S ribosome that is abundant in all bacteria and is specific for each bacterial species [11]. It interacts with translation factors during protein biosynthesis in bacteria. However, available data on the sensitivity of this kit are limited with only clinical performance trials used in the application for approval [10]. The main purpose of the present study was to determine the sensitivity of Ribotest compared with real-time polymerase chain reaction (PCR) as the gold standard. We prospectively performed these two tests simultaneously in adolescent and adult patients with CAP. In addition, we retrospectively analyzed theoretical sensitivity using M. pneumoniae PCR-positive specimens from previous studies.
http://dx.doi.org/10.1016/j.jiac.2015.02.007 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., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
2
N. Miyashita et al. / J Infect Chemother xxx (2015) 1e3
The prospective study was conducted by Kawasaki Medical School Hospital and Kawasaki Medical School Kawasaki Hospital from November 2013 to October 2014. We enrolled adolescent and adult patients with CAP. 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. Informed consent was obtained from all patients, and the study protocol was approved by the Ethics Committee of Kawasaki Medical School (approval number 1279). In the retrospective study, we used real-time PCR-positive specimens that were collected from pediatric patients with RTIs who visited 65 institutions throughout Japan, participating in the Atypical Pathogen Study Group from January 2008 to December 2013 [12,13]. A complete list of participating facilities is provided in the Appendix. Cultivation of M. pneumoniae was carried out with pleuropneumonia-like organism broth (PPLO; Oxoid, Franklin, NJ, USA) supplemented with 0.5% glucose (Wako Pure Chemicals Inc., Osaka, Japan), 20% mycoplasma supplement-G (Oxoid), and 0.0025% phenol red (Sigma-Aldrich Co. LLC., St. Louis, MO, USA) using nasopharyngeal swab specimens [14]. DNA then was extracted by using a QIAamp DNA Mini Kit (QIAGEN K. K., Tokyo, Japan) in accordance with the manufacturer's instructions. M. pneumonia DNA was detected by real-time PCR targeting a conserved part of the gene encoding the P1 adhesin gene [14]. A search for mutations at sites 2063, 2064, and 2617 in the M. pneumoniae 23S rRNA domain V gene region was performed using a direct sequencing method in isolates or samples with a positive PCR result, as reported previously [14]. Ribotest Mycoplasma was performed in accordance with the manufacturer's instructions. Antibodies to M. pneumoniae were measured using a particle agglutination test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan). During the prospective study period, 118 CAP cases were assessed using the microbiological tests. Sixteen cases were diagnosed as M. pneumoniae pneumonia; eight cases were PCR-positive, one case was culture-positive, and all cases demonstrated a fourfold increase in antibody titer. One culture-positive specimen was also PCR-positive. During the retrospective study period, 1110 cases were M. pneumoniae PCR-positive. Categorical variables of age, gender, and mutation type of the eight patients in the prospective study and the 1110 patients in retrospective study with PCR-positive M. pneumoniae infection are presented in Table 1. Among the PCR-positive cases, five cases in prospective study and 801 cases in retrospective study were found to be infected with macrolide-resistant M. pneumoniae. The correlations between Ribotest and real-time PCR results using 118 CAP cases are presented in Table 2. Ribotest was positive in 15 cases; five cases were PCR positive and 10 cases were PCR negative. When the PCR was used as the control test, the sensitivity, specificity, and overall agreement of Ribotest were 62.5%, 90.9%, and 88.9%, respectively. Among the 15 Ribotest-positive cases, six cases were serology positive and nine cases were serology negative. When the serology was used as the control test, the sensitivity, specificity, and overall agreement of Ribotest were 37.5%, 91.1%, and 83.8%, respectively. One serology positive case was observed in 10 PCR-negative and Ribotest-positive cases. Because the M. pneumoniae PCR-positive sample size in the prospective study was too small to evaluate the sensitivity of
Table 1 Categorical variables of age, gender, and mutation type of patients with PCR-positive M. pneumoniae infection in prospective and retrospective studies. Variables
Prospective study
No. of patients 8 Mean age (range), years 19.8 (16e24) No. of males/females 4/4 No. of resistant cases 5 No. of point mutation in domain V of 23S rRNA A2063Ga 5 A2063T 0 A2064G 0 A2064C 0 C2617G 0 a
Retrospective study 1110 7.6 (0e15) 544/566 801 772 20 5 3 1
A-to-G transition at position 2063 in domain V on the 23S rRNA gene.
Ribotest, we conducted the retrospective analysis using PCRpositive specimens from previous studies [12,13]. The cut-off level of Ribotest has reported as 250 copy/mL. We evaluated the detection limit of Ribotest independently in our laboratory and confirmed the same cut-off level. The swab specimens collected from patients were suspended in 3.0 mL transport medium, and then DNA was extracted using 300 mL medium. The extracted DNA was eluted in a total volume of 100 mL, and then 5 mL was used for PCR assay. Thus, the cut-off level of Ribotest was calculated as 8.3 104 copy/mL in transport medium and 2.5 105 copy/swab, respectively. Using this cut-off level, the theoretical positive number of Ribotest was calculated. Among 1110 M. pneumoniae PCR-positive specimens, 667 (60.0%) specimens were theoretically positive. In a clinical performance trial with 33 M. pneumoniae PCRpositive specimens out of 176 CAP cases, it was demonstrated that the sensitivity, specificity, and overall agreement of Ribotest compared with a PCR assay were 57.6%, 91.6%, and 85.2%, respectively [10]. These data are consistent with our present results despite the small sample size. In addition, the theoretical sensitivity with 60% of our retrospective study was also similar to the sensitivity of clinical performance trial. CAP remains a significant cause of morbidity and death worldwide. Rapid diagnostic tests are useful tools in the early presumptive diagnosis of CAP caused by certain pathogens. These results can be used to initiate appropriate antibiotic treatment and prevent antimicrobial resistance through the use of narrowspectrum antibiotics. Among the microbiologic tests, serological testing is the most common tool for the diagnosis of M. pneumoniae infection. However, this test requires paired serum samples with a 2e4 week interval and provides only a retrospective diagnosis. A rapid and simple serologic test, ImmunoCard Mycoplasma (Meridian Bioscience, Cincinnati, OH, USA), was developed for the detection of IgM antibodies [15], and this kit has become widely used in Japan. However, our study indicated that the ImmunoCard test suffers from several shortcomings. First, this kit showed many false-positive reactions in healthy adults. Second, IgM antibodies persist for several weeks and months. These findings suggest it is not best practice to base a diagnosis of M. pneumoniae pneumonia on a single assay for IgM alone [16]. Table 2 Correlations between Ribotest and real-time PCR results using 118 CAP cases in the prospective study. Real-time PCR
Ribotest Mycoplasma
Total
Positive
Negative
Positive Negative
5 3
10 100
15 103
Total
8
110
118
Please cite this article in press as: Miyashita N, et al., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
N. Miyashita et al. / J Infect Chemother xxx (2015) 1e3
In this situation, another rapid and simple kit, Ribotest Mycoplasma, became available for the diagnosis of M. pneumoniae infection. Our prospective and retrospective studies and the previous study [10] demonstrated that the diagnostic sensitivity of Ribotest was not high compared with PCR, at approximately 60%. Physicians using Ribotest should have a clear understanding of its advantages and disadvantages. It is expected that the approval of improved version of Ribotest, second generation and third generation tests. The limitation of our study was its small sample size for PCRpositive specimens. Further prospective studies are needed to clarify the sensitivity and specificity of Ribotest in patients with CAP. The results from the retrospective analysis are just supplementary support. In addition, we could not evaluate the factors affecting the results of Ribotest. In conclusion, our prospective and retrospective results demonstrated that the diagnostic sensitivity of Ribotest compared with PCR was approximately 60%. Thus, treatment decisions about M. pneumoniae pneumonia should be based on clinical findings such as Japanese Respiratory Society scoring system [4,5] and not on Ribotest results alone. Conflict of interest None. Acknowledgments This study was supported in part 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, 19-402M, 20-4030). Appendix. Participating facilities The Atypical Pathogen Study Group is formed by Hideki Asaki (Asaki Pediatric Clinic); Kazutoyo Asada (National Mie Hospital); Tomohiro Ichimaru (Saga Prefectural Hospital Koseikan); Toshio Ineda (Inada Clinic); Takuya Inoue (Chayamati Pediatric Clinic); Masakazu Umemoto (Umemoto Pediatric Clinic); Kanetsu Okura (Okura Clinic); Kenji Okada (Fukuoka National Hospital); Takashige Okada (Okada Pediatric Clinic); Teruo Okafuji (Okafuji Pediatric Clinic); Yasuko Okamoto (Okamoto Clinic); Shinichiro Oki (Higashisaga National Hospital); Keiko Oda (Kawasaki Medical School Kawasaki Hospital); Jin Ochiai (Ochiai Pediatric Clinic); Seiko Obuchi (Obuchi Clinic); Yoji Kanehara (Kanehara Pediatric Clinic); You Kanematsu (Kanematsu Pediatric Clinic); Shoji Kouno (Shomonoseki City Central Hospital); Makoto Kuramitsu (Aoba Pediatric Clinic); Katsuji Kuwakado (Kurashiki Central Hospital); Satoshi Kuwano (Kuwano Kids Clinic); Tatuso Koga (Koga Pediatric Clinic); Hayashi Komura (Komura Pediatric Clinic); Hiroshi Sakata (Asahikawa-Kosei General Hospital); Takahisa Sakuma (Sakuma Pediatric Clinic); Kazuhide Shiotsuki (Shiotsuki Internal Medicine Pediatric Clinic); Yasushi Shimada (Shimada Clinic); Makio Sugita (Kurashiki Riverside Hospital); Toru Sugimura (Sugimura Pediatric Clinic); Shumei Takeda (Takeda Pediatric Clinic); Isao Tanaka (Mizushima Central Hospital); Hiroyuki Tanaka (Tanaka Family Clinic); Naohumi Tomita (Tomita Clinic); Kensuke Nagai (Nagai Pediatric Internal Medicine Clinic); Yoshikuni Nagao (Mabi Memorial Hospital); Hidekazu Nakashima (Kojima Central Hospital); Tadashi Nagata (Nagata Pediatric Clinic); Kimiko Nakamura (Enoura Clinic);
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Kazuyo Nomura (Kama Red Cross Hospital); Kanoko Hashino (Hashino Pediatric Clinic); Yuko Hirata (Hirata Internal Medicine Pediatric Clinic); Kazumi Hiraba (Mokubo Pediatric Clinic); Takuji Fujisawa (Fujisawa Pediatric Clinic); Akiko Maki (Hashima Pediatric Clinic); Toshinobu Matsuura (Yoshino Pediatric Clinic); Nobuyoshi Mimaki (Kurashiki Medical Center); Tatsuhiko Moriguchi (Sakai Hospital Kinki University Faculty of Medicine); Shigeru Mori (Momotaro Clinic); Yoichiro Yamaguchi (Yamaguchi Pediatric Clinic); Syuji Yamada (Yamada Pediatric Clinic); Teruyo Fujimi (Fujimi Clinic); Norio Tominaga (Isahaya Health Insurance General Hospital); Syunji Hasegawa (Yamaguchi University Graduate School of Medicine); Kiyoko Nishimura (Nishimura Pediatric Clinic); Mihoko Mizuno (Daido Clinic); Jiro Iwamoto (Iizuka Hospital); Toshiyuki Iizuka (Hakuai Hospital); Shigeru Yamamoto (Daido Municipal Pediatric Clinic); Tomomichi Kurasaki (Kurosaki Pediatric Clinic); Tadashi Matsubayashi (Seirei Hamamatsu General Hospital).
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Please cite this article in press as: Miyashita N, et al., Diagnostic sensitivity of a rapid antigen test for the detection of Mycoplasma pneumoniae: Comparison with real-time PCR, J Infect Chemother (2015), http://dx.doi.org/10.1016/j.jiac.2015.02.007
