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Setting a standard for the initiation of steroid therapy in refractory or severe Mycoplasma pneumoniae pneumonia in adolescents and adults
J Infect Chemother 21 (2015) 153e160
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Original article
Setting a standard for the initiation of steroid therapy in refractory or severe Mycoplasma pneumoniae pneumonia in adolescents and adults Naoyuki Miyashita a, *, Yasuhiro Kawai a, Norikazu Inamura b, 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 22 July 2014 Received in revised form 10 October 2014 Accepted 15 October 2014 Available online 19 December 2014
Serum interleukin (IL)-18 level was thought to be a useful as a predictor of refractory or severe Mycoplasma pneumoniae pneumonia, and steroid administration is reported to be effective in this situation. The serum levels of IL-18 correlated significantly with those of lactate dehydrogenase (LDH). The purpose of this study was to set a standard for the initiation of steroid therapy in M. pneumoniae pneumonia using a simple serum marker. We analyzed 41 adolescent and adult patients with refractory or severe M. pneumoniae pneumonia who received steroid therapy, and compared them with 108 patients with M. pneumoniae pneumonia who responded to treatment promptly (control group). Serum LDH levels were significantly higher in the refractory and severe group than in the control group at the initiation of steroid therapy (723 vs 210 IU/L, respectively; p < 0.0001). From receiver operating characteristic curve analysis, we calculated serum LDH cut-off levels of 364 IU/L at initiation of steroid therapy and 302 IU/L at 1e3 days before the initiation of steroid therapy. The administration of steroids to patients in the refractory and severe group resulted in the rapid improvement of symptoms and a decrease in serum LDH levels in all patients. Serum LDH level can be used as a useful parameter to determine the initiation of steroid therapy in refractory or severe M. pneumoniae pneumonia. A serum LDH level of 302e364 IU/L seems to be an appropriate criterion for the initiation of steroid therapy. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycoplasma pneumoniae Refractory pneumonia Severe pneumonia Interleukin 18 Lactate dehydrogenase Steroid therapy
1. Introduction Mycoplasma pneumoniae is one of the major causative pathogens of community-acquired pneumonia (CAP) in children and younger adults [1]. During 2010 and 2012, large epidemics of M. pneumoniae infection occurred throughout Japan [2]. Although pneumonia due to M. pneumoniae is usually a benign, self-limited disease, some cases are known to develop into refractory or severe, life-threatening pneumonia [3e10]. The pathogenesis of refractory or severe M. pneumoniae infections is closely related to an excessive immune response against the pathogen, such as highly activated cell-mediated immune responses and vigorous expression of cytokines [1,11e15]. Thus,
* 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).
immunosuppressive therapy, such as with corticosteroids, downregulates the cell-mediated immune response and has shown a profound beneficial effect by reducing the immune-mediated pulmonary injury seen in mycoplasmal infections [3e10,14e17]. However, the appropriate timing for initiation of steroid therapy has not been investigated. Interleukin (IL)-18 is believed to be associated with the severity of pneumonia because its level increases particularly in the acute phase and it induces the expression of various cytokines [12e15]. Thus, serum IL-18 level was thought to be a useful as a predictor of refractory or severe M. pneumoniae pneumonia [12e15]. However, IL-18 cannot currently be measured in most hospitals and clinics in a timely fashion. In our preliminary study, we found a significant correlation between serum IL-18 and lactate dehydrogenase (LDH) levels [15]. The purpose of this study was to determine whether there was LDH level that could be used to determine which patients will require steroid therapy in M. pneumoniae pneumonia. We analyzed serum LDH levels consecutively in adolescent and adult patients with refractory or severe M. pneumoniae pneumonia and
http://dx.doi.org/10.1016/j.jiac.2014.10.008 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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compared them with levels in M. pneumoniae pneumonia patients who responded to antibiotic therapy promptly. 2. Materials and methods 2.1. Study population This study was conducted by 25 institutions from January 2010 to December 2013. We enrolled adolescent and adult patients with CAP who had suspected M. pneumoniae pneumonia, according to the scoring system of the Japanese Respiratory Society (JRS) CAP guidelines [18], and patients with refractory or severe CAP who were initially admitted and treated in other hospitals and transferred to our hospital before initiation of steroid therapy. 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 at Kawasaki Medical School. 2.2. Definition of refractory or severe M. pneumoniae pneumonia Refractory M. pneumoniae pneumonia was defined as follows: 1) prolonged fever for 7 days or more or 2) increasing cough and infiltrates in chest radiograph despite administration of appropriate antibiotics. Patients with severe M. pneumoniae pneumonia who required intensive care unit (ICU) admission were defined by Infectious Diseases Society of America/American Thoracic Society criteria for severe CAP [19]. The 2 major criteria-mechanical ventilation with endotracheal intubation and septic shock requiring vasopressors-are indications for admission to ICU. Patients who met the three or more of the nine minor criteriarespiratory rate 30 breaths/min, arterial oxygen pressure/fraction of inspired oxygen ratio 250, multilobar infiltrates, confusion/disorientation, Blood urea nitrogen level 20 mg/dL, white blood cell count <4000/mm3, platelet count <100,000/mm3, core temperature <36 C, and hypotension requiring aggressive fluid resuscitation-are indications for admission to ICU.
clinical symptoms and signs had not improved after 3 days minocycline administration, antibiotic therapy was changed to quinolones by the attending physicians. If minocycline was administered before admission to our hospitals, antibiotic was changed to quinolones or b-lactams plus macrolides by the attending physicians. Steroid was administered to patients who met the criteria for refractory or severe M. pneumoniae pneumonia. 2.4. Microbiological laboratory tests Microbiological tests such as Gram stain, cultures for the detection of bacteria, Legionella species, Chlamydia species, and M. pneumoniae, urinary antigen tests for the detection of Streptococcus pneumoniae and L. pneumophila and serological tests for the detection of viruses, Legionella species, Chlamydia species, Coxiella burnetii, and M. pneumoniae were performed as described previously [20,21]. Cultivation of M. pneumoniae was carried out using pleuropneumonia-like organism broth (Difco, Detroit, MI, USA). 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 polymerase chain reaction (PCR) targeting a conserved part of the gene encoding P1 adhesin. Antibodies to M. pneumoniae were measured using a particle agglutination test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan). The microbial etiology was classified as “definitive”, “presumptive”, or “unknown” as described previously [20,21]. 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 samples with a positive culture or PCR result and the minimum inhibitory concentration (MIC) of 11 antimicrobial agents for the M. pneumoniae isolates was determined by micro-dilution methods as described previously [22,23]. 2.5. Statistical analysis Statistical analysis was performed using Stat View version 5.0. (SAS Institute Inc, Cary, NC, USA). The incidence of underlying conditions was analyzed using Fisher's exact test. Serum LDH values are presented as the median ± interquartile range. Mean age of patients and laboratory data were compared using ManneWhitney's U test. Serum LDH cut-off level for the initiation of steroid therapy was calculated by employing a receiver operating characteristic (ROC) curve [24].
2.3. Study protocol 3. Results Nasopharyngeal swab or sputum specimens, serum samples, and urine were collected for microbiological tests at admission. Bronchoscopic examination was employed to obtain specimens in patients with endotracheal intubation. Blood tests for peripheral white blood cell count, C-reactive protein, total protein, LDH, alanine aminotransferase, and aspartate aminotransferase were performed on admission, at 2e4 days after antibiotic therapy, at 5e7 days after antibiotic therapy, at the end of antibiotic therapy, and at 30 days after antibiotic therapy. The serum level of IL-18 was measured on admission with a Human IL-18 ELISA kit (MBL Co. Ltd, Nagoya, Japan) according to the supplier's instructions. Clinical information including laboratory data in patients with refractory or severe M. pneumoniae pneumonia before initiation of steroid therapy was collected from former hospitals. Blood tests including LDH were performed in all patients at 1e3 days and 4e7 days before steroid therapy. After admission, minocycline was administered intravenously twice daily at doses of 100 mg. If adverse events were observed or
3.1. Patient characteristics Cases of M. pneumoniae pneumonia mixed with other microorganisms were excluded in this study. During the study period, 108 CAP cases who met all parameters out of six of the JRS scoring system [18] were assessed using the microbiological tests described in the Materials and Methods. The diagnostic sensitivity and specificity of the JRS scoring system for the presumptive diagnosis of M. pneumoniae pneumonia were 28e35% and 99e100%, respectively [18,20,21]. All CAP cases were diagnosed as M. pneumoniae pneumonia; fifty-four cases were PCR positive, 10 cases were culture positive, and 106 cases demonstrated a four-fold increase in antibody titer. All 108 cases of M. pneumoniae pneumonia were responded to minocycline promptly and no relapse was observed after 5e7 days minocycline administration. Finally, we analyzed 108 cases of M. pneumoniae pneumonia as a control group.
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correlation coefficient. The laboratory data were used at the initiation of steroid therapy in 41 patients in the refractory and severe group. Significant correlations were not found between serum levels of IL-18 and white blood cells, C-reactive protein, total protein, alanine aminotransferase, or aspartate aminotransferase values. Only the correlation between IL-18 and LDH was statistically significant in both control group (r2 ¼ 0.4677, p < 0.001, Fig. 1) and refractory and severe group (r2 ¼ 0.5145, p < 0.001, Fig. 2).
Among the refractory or severe CAP cases who transferred to our hospital, 41 cases were diagnosed as M. pneumoniae pneumonia. Twenty cases were PCR positive, 4 cases were culture positive, and all cases demonstrated a four-fold increase in antibody titer. These 41 cases were divided into three groups; refractory group (n ¼ 11), severe group who required mechanical ventilation (n ¼ 19), and severe group who did not required mechanical ventilation (n ¼ 11). All patients presented acute respiratory failure with arterial oxygen pressure/fraction of inspired oxygen ratio 250 and respiratory rate 30 breaths/min at the admission to our hospitals. Categorical variables of age, gender, co-morbid illnesses, mutation type, and prior prescription of antibiotics before admission to the study hospital of 149 patients with M. pneumoniae pneumonia are presented in Table 1. No significant difference was observed in mean age, gender, or number of co-morbid illnesses among the four groups.
3.4. Change in serum LDH levels in each group In the control group, 22 patients (20%) had elevated serum LDH levels (normal range, 120e240 IU/L), and the median serum LDH level decreased after appropriate antibiotic therapy (Fig. 3). In the refractory or severe groups, the median serum LDH level clearly increased after the onset of symptoms and decreased after steroid therapy (Figs. 4e6). At the initiation of steroid therapy, the median serum LDH level was significantly higher in the refractory group (701 IU/L), severe with mechanical ventilation group (692 IU/L), and severe without mechanical ventilation group (766 IU/L) than in the control group (210 IU/L, p < 0.0001).
3.2. Detection of macrolide-resistant M. pneumoniae Among the 74 culture- and/or PCR-positive cases, 48 cases were found to be infected with macrolide-resistant M. pneumoniae (Table 1). Forty-seven had an A-to-G transition at position 2063 in domain V on the 23S rRNA gene (A2063G) and one had an A-to-G transition at position 2064 (A2064G). No mutations at site 2617 in domain V of the 23S rRNA gene were observed. Among eight of 14 isolates that had a macrolide-resistance mutation, the MIC50 values for 14- and 15-membered macrolides, such as erythromycin, clarithromycin, and azithromycin were >128 mg/ml, >128 mg/ml, and 128 mg/ml, respectively; MIC values in the macrolide-sensitive reference strain were 0.0039 mg/ml, 0.002 mg/ml, and 0.00025 mg/ml, respectively. Conversely, tetracycline, minocycline, and all four quinolones showed good antimycoplasmal activity, with MIC50 values of 0.5 mg/ml, 2.0 mg/ml, and 0.5e0.0625 mg/ml, respectively, against macrolide-resistant M. pneumoniae isolates, which were equal to those for the macrolide-sensitive reference strain.
3.5. Cut-off level of serum LDH The serum LDH cut-off level for the initiation of steroid therapy was calculated by employing an ROC curve. The LDH levels were used at admission for patients in the control group and at initiation, at 1e3 days before, and at 4e7 days before steroid therapy in patients in the refractory or severe group. The serum LDH cut-off level suggested was similar among refractory and severe groups and ranged 364e395 IU/L at the initiation of steroid therapy, 302e363 IU/L at 1e3 days before steroid therapy, and 243e264 IU/L at 4e7 days before steroid therapy (Table 2). 3.6. Discrepancy cases Among the refractory and severe groups, one case (21-year-old female) showed low LDH level, 280 IU/L, at the initiation of steroid therapy. She visited our hospital increasing of cough, productive sputum, and dyspnea despite receiving antibiotic in other clinic from 4 days before. She met the three minor criteria of severe CAP
3.3. Relationship between IL-18 levels and other clinical laboratory data The association between serum IL-18 levels and other clinical laboratory data was evaluated using the Spearman's rank
Table 1 Categorical variables age, gender, co-morbid illness, mutation type, and prior prescription of antibiotics before admission to the study hospitals of 149 patients with M. pneumoniae pneumonia. Variables
Control group
Refractory group
Severe group with mechanical ventilation
Severe group without mechanical ventilation
No. of patients Mean age (Range), years No. of males/females No. of patients with co-morbid illness No. of patients with confounding factors for elevation of serum LDH level except for M. pneumoniae infectiona No. of resistant cases/M. pneumoniae PCR or culture positive cases No. of point mutation in domain V of 23S rRNA
108 30.3 ± 12.6 (16e75) 48/60 15 3
11 35.7 ± 9.3 (16e49) 5/6 1 0
19 35.7 ± 10.7 (16e51) 7/12 4 0
11 31.5 ± 13.4 (16e60) 5/6 1 0
36/54
1/3
7/11
4/6
A2063G 35 A2064G 1
A2063G 1
A2063G 7
A2063G 4
16 9 0 0 5
9 4 6 3 4
19 2 1 5 2
11 1 1 3 1
No. of patients with prior prescription before admission to study hospitalsb b-lactams Macrolides Minocycline Clindamycin Quinolones
a Confounding factors including liver disease, heart disease, renal disease, skeletal muscle disease, hematologic disease, pulmonary thromboembolism, neoplastic disease and infection. b Several patients with refractory or severe pneumonia received one or more antibiotics.
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Fig. 1. Correlation between serum levels of IL-18 and LDH in patients with control group at the initiation of the appropriate antibiotics. A significant correlation was found between serum levels of IL-18 and LDH.
Fig. 2. Correlation between serum levels of IL-18 and LDH in patients with refractory and severe group at the initiation of steroid therapy. A significant correlation was found between serum levels of IL-18 and LDH.
[19] and M. pneumoniae PCR was positive. Then, she admitted to our hospital and received minocycline and steroid therapy. This case was diagnosed as rapid progress type of M. pneumoniae pneumonia. Among the control group, one case (37-year-old male) showed high LDH level, 391 IU/L, at the initiation of appropriate antibiotic therapy. This case was occurred within family outbreak and he visited our hospital complaining of cough, dyspnea and fever for 10 days. At that time, he had hypoxemia but met one minor criteria of severe CAP [19]. This case was diagnosed as M. pneumoniae pneumonia with acute respiratory failure.
3.7. Clinical course in refractory and severe patients Methylprednisolone pulse therapy at a dose of 1 g/day for 3 days was administered to all refractory and severe patients. Then oral prednisolone 40 mg/day was administered and weaned over 1e2 weeks. After steroid administration, the clinical and radiographic findings of all patients improved rapidly, and serum LDH levels decreased to the normal range. No refractory and severe patients had worsening of clinical and radiographic findings after discontinuation of prednisolone, and no patient had further complications
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Fig. 3. Changes in serum LDH levels in 108 control patients with M. pneumoniae pneumonia at four points before and after antibiotic treatment. Bars indicate range and squares indicate median value ± interquartile range.
associated with infection. No adverse events were reported during or after steroid therapy. 4. Discussion Several studies, including our results, demonstrated that an excessive host-cellular response, especially IL-18, plays a critical role in the development of serious pulmonary manifestations [12e15], and serum levels of IL-18 were significantly correlated
with those of LDH [14,15]. Because the measurement of serum IL-18 levels cannot be always performed, LDH instead of IL-18 was thought to be useful as a predictor of refractory or severe M. pneumoniae pneumonia in the daily clinical setting. Therefore, in this study we analyzed serum LDH levels consecutively in order to set a standard for the initiation of steroid therapy in M. pneumoniae pneumonia. In our preliminary study among pediatric patients, we demonstrated that steroid administration would be required when serum LDH levels are 410 IU/L [15]. However, the sample size was
Fig. 4. Changes in serum LDH levels in 11 patients with refractory M. pneumoniae pneumonia at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
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Fig. 5. Changes in serum LDH levels in 19 patients with severe M. pneumoniae pneumonia who required mechanical ventilation at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
very small (n ¼ 5) to determine the appropriate criterion for the initiation of steroid therapy. In this study, our results suggested that the serum LDH cut-off level was 364 IU/L at initiation of steroid therapy and 302 IU/L at 1e3 days before the initiation of steroid therapy in patients with refractory and severe M. pneumoniae pneumonia. To prevent the progression of pneumonia severity, serum LDH levels of 302e364 IU/L seemed to be an appropriate criterion for the initiation of steroid therapy. Our results also demonstrated that steroids combined with appropriate antibiotic therapy reduced serum LDH levels in the
refractory and severe groups to the same level as in the control group (Figs. 3e6). Thus, serum LDH levels may be a useful marker for the evaluation of therapeutic efficacy in refractory and severe M. pneumoniae pneumonia. Histopathologically, M. pneumoniae pneumonia is characterized by acute cellular bronchiolitis and by peribronchial and perivascular interstitial opacities. These histopathological findings closely reflect chest HRCT findings, which indicate that the combination of bronchial wall thickening and centrilobular nodules [25]. However, at the progressed stage refractory and severe
Fig. 6. Changes in serum LDH levels in 11 patients with severe M. pneumoniae pneumonia who did not required mechanical ventilation at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
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Table 2 Serum LDH cut-off value, sensitivity, specificity, and area under the receiver operating characteristic curve for the initiation of steroid therapy in patients with refractory and severe M. pneumoniae pneumonia. Variables At the initiation of steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients At 1e3 days before steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients At 4e7 days before steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients
LDH cut-off value (IU/L)
Sensitivity (%)
Specificity (%)
Area under the ROC curve
395 367 364 364
100 89.5 90.9 92.7
100 99.1 99.1 99.1
1 0.992 0.989 0.993
302 363 312 302
100 84.2 90.9 92.7
92.6 99.1 92.6 92.6
0.984 0.983 0.981 0.983
258 264 243 243
63.3 63.2 72.7 70.7
84.3 87.0 79.6 79.6
0.769 0.808 0.808 0.804
LDH, lactate dehydrogenase; ROC, receiver operating characteristic.
patients showed bilateral diffuse infiltrates and did not show the radiographic features of M. pneumoniae pneumonia that was observed in the non-progressed stage (control group). Because of the non-specific radiographic findings, chest HRCT findings thought to be not useful parameter to determine the point to initiate steroid therapy in refractory or severe M. pneumoniae pneumonia. Before initiating steroid therapy in CAP patients, we must be define or suspect M. pneumoniae infection, because there are no evidence to support the well tolerated use of systemic steroids in CAP due to other pathogens [26e28]. Among CAP pathogens, we have demonstrated that the presumptive diagnosis of M. pneumoniae pneumonia is relatively easy using the combination of three rapid and simple diagnostic methods: the JRS scoring system, chest computed tomography, and ImmunoCard Mycoplasma [20e22]. In addition, two commercially available point-ofcare test kits for detection of the M. pneumoniae antigen, Ribotest
Community-acquired pneumonia patients with: 1. met all parameters out of six of the JRS scoring system or 2. positive for M. pneumoniae rapid antigen tests or 3. met four or more parameters out of six of the JRS scoring system + chest CT findings
Initiation of anti-M. pneumoniae antibiotics
Deterioration of clinical and radiographic findings
Check for serum LDH level consecutively
Serum LDH level: 302-363 IU/L
Serum LDH level: ≥ 364 IU/L
Consider to initiation of steroid therapy
Initiation of steroid therapy
Fig. 7. A stepwise algorithm to correlate diagnosis, serum markers, and initiation of steroid therapy in M. pneumoniae pneumonia.
Mycoplasma (Asahikasei Pharma Co., Tokyo, Japan) and PrimeCheck Mycoplasma Antigen (Alfresa Pharma Co., Osaka, Japan) have been available since 2013 in Japan [29]. Using the combination of these five rapid and simple methods, the diagnostic sensitivity and specificity of M. pneumoniae pneumonia have increased. A stepwise algorithm to correlate diagnosis, serum markers, and initiation of steroid therapy is summarized in Fig. 7. M. pneumoniae showing resistance to macrolides was isolated from patients with CAP in 2000, and macrolide resistance has become widespread in Japan [22,23]. In our previous studies, no significant differences in pneumonia severity were observed between macrolide-resistant patients and macrolide-sensitive patients [22,23]. In this study, the prevalence of macrolide-resistant strain was almost identical among control and severe groups. Thus, macrolide-resistant strain seems to be not related with pneumonia severity. Pulmonary injury associated with refractory or severe M. pneumoniae pneumonia thought to be due to an excessive, activated cell-mediated immune response to pathogens rather than to direct microbial damage [1,13]. If refractory or severe M. pneumoniae pneumonia is an immune mediated disease, immune suppressive therapy is a rational approach. In our study, corticosteroid combined with antibiotics therapy demonstrated the satisfactory clinical effects and reduced serum IL-18 and LDH levels in patients with refractory or severe M. pneumoniae pneumonia [3,15]. The use of corticosteroids combined with antibiotics therapy for decreasing the inflammatory response with dramatic beneficial effect has also been demonstrated in several studies [4e10,14,16,17]. Thus, high level of serum IL-18 and LDH levels in refractory or severe group may be due to host immune response rather than to virulence of M. pneumoniae. However, no available data for explanation of mechanism in which linked the raised serum IL-18 and LDH levels in refractory or severe group. Further study is needed. Our study had several limitations. Since present study was not case-control study, our study lacks the data of explaining the benefits of using steroid for the patients with refractory or severe M. pneumoniae pneumonia who had high value of serum LDH. Further prospective studies are needed to confirm the beneficial effect of steroids in patients with high value of serum LDH. Second, we could not demonstrate the presence of macrolide-resistant mutations in all patients. Thus, in this study, we selected minocycline or quinolones as antibiotics, because these agents are clinically and bacteriologically more effective than macrolides [22,23]. The change in serum LDH levels in patients with M. pneumoniae
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pneumonia in both macrolide-resistant and macrolide-sensitive strains who were treated with macrolides should be investigated. Third, it has been suggested that elevated serum ferritin and urine b2-microglobulin levels, which suggest complications of hypercytokinemic conditions, may be associated with the severity of pneumonia [6]. In addition, the strong correlation between serum cortisol and adrenocorticotropic hormone levels and pneumonia severity has been reported [30]. In this study, however, we did not evaluate these markers because the results cannot be obtained promptly. Finally, we enrolled only CAP cases who met all parameters out of six of the JRS scoring system because of the selection of definitive cases with M. pneumoniae pneumonia exactly. Thus, we missed many cases with M. pneumoniae pneumonia during the study period. The number of patients including refractory or severe cases reflects selection bias. In conclusion, our results suggest that the serum LDH level can be a useful parameter to determine the point to initiate steroid therapy in refractory or severe M. pneumoniae pneumonia. A serum LDH level of 302e364 IU/L seemed to be an appropriate criterion for the initiation of steroid therapy. In addition, serum LDH level seems to be a useful marker for the evaluation of therapeutic efficacy. 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, 19402M, 20-4030). Appendix This study was conducted by following institutions; Kato T. (Asahigaoka Hospital); Yamada H. (Takahashi Central Hospital); Kawasaki F. (Okayama Saidaiji Hospital); Tanaka H. (Saijo Aijukai Hospital); Okimoto N. (Satou Memorial Hospital); Masuda T. (Tamano Central Hospital); Nakatani S. (Nakatani Geka Hospital); Maruyama T. (Mie National Hospital); Nakahama C. (Nakahama Clinic); Yoneyama H. and Nakamura J. (Kasaoka Daiichi Hospital); Kurihara T. (Kawasaki Hospital); Kawanishi M. (Kaneda Hospital); Kimura M. (Kimura Clinic); Hara H. and Matsushima T. (Kurashiki Daiichi Hospital); Yano T. (Kurashiki Memorial Hospital); Yagi S. (Heisei Hospital); Ikeda M. (Kojima Central Hospital); Mouri K. (Tamashima Central Hospital); Kato T. (Nakashima Hospital); Sugimura S. (Okayama Kyoritsu General Hospital); Doi M. and Kuwabara M. (Kenritsu Hiroshima Hospital); Hashiguchi K. (Nagasaki Genbaku Hospital); and Nagatomo Y. and Okayama A. (University of Miyazaki). References [1] Waites KB, Talkington DF. Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 2004;17:697e728. [2] National Institute of Health. Infectious Disease Surveillance Center. Mycoplasma pneumoniae pneumonia. http://idsc.nih.go.jp/idwr/kanja/ weeklygraph/18myco.html. [3] 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. [4] 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.
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Clinical implications of interleukin-18 levels in pediatric patients with Mycoplasma pneumoniae pneumonia. J Infect Chemother 2011;17:803e6. [15] Inamura M, Miyashita N, Hasegawa S, Kato A, Fukuda Y, Saitoh A, et al. Management of refractory Mycoplasma pneumoniae pneumonia: utility of measuring serum lactate dehydrogenase level. J Infect Chemother 2014;20: 270e3. [16] Tagliabue C, Salvatore CM, Techasaensiri C, Mejias A, Torres JP, Katz K, et al. The impact of steroids given with macrolide therapy on experimental Mycoplasma pneumoniae respiratory infection. J Infect Dis 2008;198:1180e8. [17] Hirano S, Wada H, Nakagaki K, Saraya T, Kurai D, Mikura S, et al. Inflammation provoked Mycoplasma pneumoniae exact: implications for combination treatment with clarithromycin and dexamethasone. FEMS Immunol Med Microbiol 2011;62:182e9. [18] 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;11:79e133. [19] Mandell LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell GD, Dean NC, et al., American Thoracic Society. Infectious Diseases Society of America/ American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007;44:S27e72. [20] 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. [21] 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. [22] 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. [23] 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. [24] Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982;143:29e36. [25] Miyashita N, Sugiu T, Kawai K, Oda K, Yamaguchi T, Ouchi K, et al. Radiographic features of Mycoplasma pneumoniae pneumonia: differential diagnosis and performance timing. BMC Med Imaging 2009;9:7. [26] Principi N, Bianchini S, Baggi E, Esposito S. No evidence for the effectiveness of systemic corticosteroids in acute pharyngitis, community-acquired pneumonia and acute otitis media. Eur J Clin Microbiol Infect Dis 2013;32:151e60. [27] Shafiq M, Mansoor MS, Khan AA, Sohail MR, Murad MH. Adjuvant steroid therapy in community-acquired pneumonia: a systemic review and metaanalysis. J Hosp Med 2013;8:68e75. [28] Povoa P, Salluh JI. What is the role of steroids in pneumonia therapy? Curr Opin Infect Dis 2012;25:199e204. [29] Sawa T, Kimura S, Hosono-Honda N, Fujita K, Yoshizawa S, Harada Y, et al. Diagnostic usefulness of ribosomal protein L7/L12 for pneumococcal pneumonia in a mouse model. J Clin Microbiol 2013;51:70e6. [30] Gotoh S, Nishimura N, Takahashi O, Shiratsuka H, Horinouchi H, Ono H, et al. Adrenal function in patients with community-acquired pneumonia. Eur Respir J 2008;31:1269e73.
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Original article
Setting a standard for the initiation of steroid therapy in refractory or severe Mycoplasma pneumoniae pneumonia in adolescents and adults Naoyuki Miyashita a, *, Yasuhiro Kawai a, Norikazu Inamura b, 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 22 July 2014 Received in revised form 10 October 2014 Accepted 15 October 2014 Available online 19 December 2014
Serum interleukin (IL)-18 level was thought to be a useful as a predictor of refractory or severe Mycoplasma pneumoniae pneumonia, and steroid administration is reported to be effective in this situation. The serum levels of IL-18 correlated significantly with those of lactate dehydrogenase (LDH). The purpose of this study was to set a standard for the initiation of steroid therapy in M. pneumoniae pneumonia using a simple serum marker. We analyzed 41 adolescent and adult patients with refractory or severe M. pneumoniae pneumonia who received steroid therapy, and compared them with 108 patients with M. pneumoniae pneumonia who responded to treatment promptly (control group). Serum LDH levels were significantly higher in the refractory and severe group than in the control group at the initiation of steroid therapy (723 vs 210 IU/L, respectively; p < 0.0001). From receiver operating characteristic curve analysis, we calculated serum LDH cut-off levels of 364 IU/L at initiation of steroid therapy and 302 IU/L at 1e3 days before the initiation of steroid therapy. The administration of steroids to patients in the refractory and severe group resulted in the rapid improvement of symptoms and a decrease in serum LDH levels in all patients. Serum LDH level can be used as a useful parameter to determine the initiation of steroid therapy in refractory or severe M. pneumoniae pneumonia. A serum LDH level of 302e364 IU/L seems to be an appropriate criterion for the initiation of steroid therapy. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Mycoplasma pneumoniae Refractory pneumonia Severe pneumonia Interleukin 18 Lactate dehydrogenase Steroid therapy
1. Introduction Mycoplasma pneumoniae is one of the major causative pathogens of community-acquired pneumonia (CAP) in children and younger adults [1]. During 2010 and 2012, large epidemics of M. pneumoniae infection occurred throughout Japan [2]. Although pneumonia due to M. pneumoniae is usually a benign, self-limited disease, some cases are known to develop into refractory or severe, life-threatening pneumonia [3e10]. The pathogenesis of refractory or severe M. pneumoniae infections is closely related to an excessive immune response against the pathogen, such as highly activated cell-mediated immune responses and vigorous expression of cytokines [1,11e15]. Thus,
* 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).
immunosuppressive therapy, such as with corticosteroids, downregulates the cell-mediated immune response and has shown a profound beneficial effect by reducing the immune-mediated pulmonary injury seen in mycoplasmal infections [3e10,14e17]. However, the appropriate timing for initiation of steroid therapy has not been investigated. Interleukin (IL)-18 is believed to be associated with the severity of pneumonia because its level increases particularly in the acute phase and it induces the expression of various cytokines [12e15]. Thus, serum IL-18 level was thought to be a useful as a predictor of refractory or severe M. pneumoniae pneumonia [12e15]. However, IL-18 cannot currently be measured in most hospitals and clinics in a timely fashion. In our preliminary study, we found a significant correlation between serum IL-18 and lactate dehydrogenase (LDH) levels [15]. The purpose of this study was to determine whether there was LDH level that could be used to determine which patients will require steroid therapy in M. pneumoniae pneumonia. We analyzed serum LDH levels consecutively in adolescent and adult patients with refractory or severe M. pneumoniae pneumonia and
http://dx.doi.org/10.1016/j.jiac.2014.10.008 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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compared them with levels in M. pneumoniae pneumonia patients who responded to antibiotic therapy promptly. 2. Materials and methods 2.1. Study population This study was conducted by 25 institutions from January 2010 to December 2013. We enrolled adolescent and adult patients with CAP who had suspected M. pneumoniae pneumonia, according to the scoring system of the Japanese Respiratory Society (JRS) CAP guidelines [18], and patients with refractory or severe CAP who were initially admitted and treated in other hospitals and transferred to our hospital before initiation of steroid therapy. 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 at Kawasaki Medical School. 2.2. Definition of refractory or severe M. pneumoniae pneumonia Refractory M. pneumoniae pneumonia was defined as follows: 1) prolonged fever for 7 days or more or 2) increasing cough and infiltrates in chest radiograph despite administration of appropriate antibiotics. Patients with severe M. pneumoniae pneumonia who required intensive care unit (ICU) admission were defined by Infectious Diseases Society of America/American Thoracic Society criteria for severe CAP [19]. The 2 major criteria-mechanical ventilation with endotracheal intubation and septic shock requiring vasopressors-are indications for admission to ICU. Patients who met the three or more of the nine minor criteriarespiratory rate 30 breaths/min, arterial oxygen pressure/fraction of inspired oxygen ratio 250, multilobar infiltrates, confusion/disorientation, Blood urea nitrogen level 20 mg/dL, white blood cell count <4000/mm3, platelet count <100,000/mm3, core temperature <36 C, and hypotension requiring aggressive fluid resuscitation-are indications for admission to ICU.
clinical symptoms and signs had not improved after 3 days minocycline administration, antibiotic therapy was changed to quinolones by the attending physicians. If minocycline was administered before admission to our hospitals, antibiotic was changed to quinolones or b-lactams plus macrolides by the attending physicians. Steroid was administered to patients who met the criteria for refractory or severe M. pneumoniae pneumonia. 2.4. Microbiological laboratory tests Microbiological tests such as Gram stain, cultures for the detection of bacteria, Legionella species, Chlamydia species, and M. pneumoniae, urinary antigen tests for the detection of Streptococcus pneumoniae and L. pneumophila and serological tests for the detection of viruses, Legionella species, Chlamydia species, Coxiella burnetii, and M. pneumoniae were performed as described previously [20,21]. Cultivation of M. pneumoniae was carried out using pleuropneumonia-like organism broth (Difco, Detroit, MI, USA). 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 polymerase chain reaction (PCR) targeting a conserved part of the gene encoding P1 adhesin. Antibodies to M. pneumoniae were measured using a particle agglutination test (Serodia-Myco II kit, Fujirebio, Tokyo, Japan). The microbial etiology was classified as “definitive”, “presumptive”, or “unknown” as described previously [20,21]. 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 samples with a positive culture or PCR result and the minimum inhibitory concentration (MIC) of 11 antimicrobial agents for the M. pneumoniae isolates was determined by micro-dilution methods as described previously [22,23]. 2.5. Statistical analysis Statistical analysis was performed using Stat View version 5.0. (SAS Institute Inc, Cary, NC, USA). The incidence of underlying conditions was analyzed using Fisher's exact test. Serum LDH values are presented as the median ± interquartile range. Mean age of patients and laboratory data were compared using ManneWhitney's U test. Serum LDH cut-off level for the initiation of steroid therapy was calculated by employing a receiver operating characteristic (ROC) curve [24].
2.3. Study protocol 3. Results Nasopharyngeal swab or sputum specimens, serum samples, and urine were collected for microbiological tests at admission. Bronchoscopic examination was employed to obtain specimens in patients with endotracheal intubation. Blood tests for peripheral white blood cell count, C-reactive protein, total protein, LDH, alanine aminotransferase, and aspartate aminotransferase were performed on admission, at 2e4 days after antibiotic therapy, at 5e7 days after antibiotic therapy, at the end of antibiotic therapy, and at 30 days after antibiotic therapy. The serum level of IL-18 was measured on admission with a Human IL-18 ELISA kit (MBL Co. Ltd, Nagoya, Japan) according to the supplier's instructions. Clinical information including laboratory data in patients with refractory or severe M. pneumoniae pneumonia before initiation of steroid therapy was collected from former hospitals. Blood tests including LDH were performed in all patients at 1e3 days and 4e7 days before steroid therapy. After admission, minocycline was administered intravenously twice daily at doses of 100 mg. If adverse events were observed or
3.1. Patient characteristics Cases of M. pneumoniae pneumonia mixed with other microorganisms were excluded in this study. During the study period, 108 CAP cases who met all parameters out of six of the JRS scoring system [18] were assessed using the microbiological tests described in the Materials and Methods. The diagnostic sensitivity and specificity of the JRS scoring system for the presumptive diagnosis of M. pneumoniae pneumonia were 28e35% and 99e100%, respectively [18,20,21]. All CAP cases were diagnosed as M. pneumoniae pneumonia; fifty-four cases were PCR positive, 10 cases were culture positive, and 106 cases demonstrated a four-fold increase in antibody titer. All 108 cases of M. pneumoniae pneumonia were responded to minocycline promptly and no relapse was observed after 5e7 days minocycline administration. Finally, we analyzed 108 cases of M. pneumoniae pneumonia as a control group.
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correlation coefficient. The laboratory data were used at the initiation of steroid therapy in 41 patients in the refractory and severe group. Significant correlations were not found between serum levels of IL-18 and white blood cells, C-reactive protein, total protein, alanine aminotransferase, or aspartate aminotransferase values. Only the correlation between IL-18 and LDH was statistically significant in both control group (r2 ¼ 0.4677, p < 0.001, Fig. 1) and refractory and severe group (r2 ¼ 0.5145, p < 0.001, Fig. 2).
Among the refractory or severe CAP cases who transferred to our hospital, 41 cases were diagnosed as M. pneumoniae pneumonia. Twenty cases were PCR positive, 4 cases were culture positive, and all cases demonstrated a four-fold increase in antibody titer. These 41 cases were divided into three groups; refractory group (n ¼ 11), severe group who required mechanical ventilation (n ¼ 19), and severe group who did not required mechanical ventilation (n ¼ 11). All patients presented acute respiratory failure with arterial oxygen pressure/fraction of inspired oxygen ratio 250 and respiratory rate 30 breaths/min at the admission to our hospitals. Categorical variables of age, gender, co-morbid illnesses, mutation type, and prior prescription of antibiotics before admission to the study hospital of 149 patients with M. pneumoniae pneumonia are presented in Table 1. No significant difference was observed in mean age, gender, or number of co-morbid illnesses among the four groups.
3.4. Change in serum LDH levels in each group In the control group, 22 patients (20%) had elevated serum LDH levels (normal range, 120e240 IU/L), and the median serum LDH level decreased after appropriate antibiotic therapy (Fig. 3). In the refractory or severe groups, the median serum LDH level clearly increased after the onset of symptoms and decreased after steroid therapy (Figs. 4e6). At the initiation of steroid therapy, the median serum LDH level was significantly higher in the refractory group (701 IU/L), severe with mechanical ventilation group (692 IU/L), and severe without mechanical ventilation group (766 IU/L) than in the control group (210 IU/L, p < 0.0001).
3.2. Detection of macrolide-resistant M. pneumoniae Among the 74 culture- and/or PCR-positive cases, 48 cases were found to be infected with macrolide-resistant M. pneumoniae (Table 1). Forty-seven had an A-to-G transition at position 2063 in domain V on the 23S rRNA gene (A2063G) and one had an A-to-G transition at position 2064 (A2064G). No mutations at site 2617 in domain V of the 23S rRNA gene were observed. Among eight of 14 isolates that had a macrolide-resistance mutation, the MIC50 values for 14- and 15-membered macrolides, such as erythromycin, clarithromycin, and azithromycin were >128 mg/ml, >128 mg/ml, and 128 mg/ml, respectively; MIC values in the macrolide-sensitive reference strain were 0.0039 mg/ml, 0.002 mg/ml, and 0.00025 mg/ml, respectively. Conversely, tetracycline, minocycline, and all four quinolones showed good antimycoplasmal activity, with MIC50 values of 0.5 mg/ml, 2.0 mg/ml, and 0.5e0.0625 mg/ml, respectively, against macrolide-resistant M. pneumoniae isolates, which were equal to those for the macrolide-sensitive reference strain.
3.5. Cut-off level of serum LDH The serum LDH cut-off level for the initiation of steroid therapy was calculated by employing an ROC curve. The LDH levels were used at admission for patients in the control group and at initiation, at 1e3 days before, and at 4e7 days before steroid therapy in patients in the refractory or severe group. The serum LDH cut-off level suggested was similar among refractory and severe groups and ranged 364e395 IU/L at the initiation of steroid therapy, 302e363 IU/L at 1e3 days before steroid therapy, and 243e264 IU/L at 4e7 days before steroid therapy (Table 2). 3.6. Discrepancy cases Among the refractory and severe groups, one case (21-year-old female) showed low LDH level, 280 IU/L, at the initiation of steroid therapy. She visited our hospital increasing of cough, productive sputum, and dyspnea despite receiving antibiotic in other clinic from 4 days before. She met the three minor criteria of severe CAP
3.3. Relationship between IL-18 levels and other clinical laboratory data The association between serum IL-18 levels and other clinical laboratory data was evaluated using the Spearman's rank
Table 1 Categorical variables age, gender, co-morbid illness, mutation type, and prior prescription of antibiotics before admission to the study hospitals of 149 patients with M. pneumoniae pneumonia. Variables
Control group
Refractory group
Severe group with mechanical ventilation
Severe group without mechanical ventilation
No. of patients Mean age (Range), years No. of males/females No. of patients with co-morbid illness No. of patients with confounding factors for elevation of serum LDH level except for M. pneumoniae infectiona No. of resistant cases/M. pneumoniae PCR or culture positive cases No. of point mutation in domain V of 23S rRNA
108 30.3 ± 12.6 (16e75) 48/60 15 3
11 35.7 ± 9.3 (16e49) 5/6 1 0
19 35.7 ± 10.7 (16e51) 7/12 4 0
11 31.5 ± 13.4 (16e60) 5/6 1 0
36/54
1/3
7/11
4/6
A2063G 35 A2064G 1
A2063G 1
A2063G 7
A2063G 4
16 9 0 0 5
9 4 6 3 4
19 2 1 5 2
11 1 1 3 1
No. of patients with prior prescription before admission to study hospitalsb b-lactams Macrolides Minocycline Clindamycin Quinolones
a Confounding factors including liver disease, heart disease, renal disease, skeletal muscle disease, hematologic disease, pulmonary thromboembolism, neoplastic disease and infection. b Several patients with refractory or severe pneumonia received one or more antibiotics.
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Fig. 1. Correlation between serum levels of IL-18 and LDH in patients with control group at the initiation of the appropriate antibiotics. A significant correlation was found between serum levels of IL-18 and LDH.
Fig. 2. Correlation between serum levels of IL-18 and LDH in patients with refractory and severe group at the initiation of steroid therapy. A significant correlation was found between serum levels of IL-18 and LDH.
[19] and M. pneumoniae PCR was positive. Then, she admitted to our hospital and received minocycline and steroid therapy. This case was diagnosed as rapid progress type of M. pneumoniae pneumonia. Among the control group, one case (37-year-old male) showed high LDH level, 391 IU/L, at the initiation of appropriate antibiotic therapy. This case was occurred within family outbreak and he visited our hospital complaining of cough, dyspnea and fever for 10 days. At that time, he had hypoxemia but met one minor criteria of severe CAP [19]. This case was diagnosed as M. pneumoniae pneumonia with acute respiratory failure.
3.7. Clinical course in refractory and severe patients Methylprednisolone pulse therapy at a dose of 1 g/day for 3 days was administered to all refractory and severe patients. Then oral prednisolone 40 mg/day was administered and weaned over 1e2 weeks. After steroid administration, the clinical and radiographic findings of all patients improved rapidly, and serum LDH levels decreased to the normal range. No refractory and severe patients had worsening of clinical and radiographic findings after discontinuation of prednisolone, and no patient had further complications
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Fig. 3. Changes in serum LDH levels in 108 control patients with M. pneumoniae pneumonia at four points before and after antibiotic treatment. Bars indicate range and squares indicate median value ± interquartile range.
associated with infection. No adverse events were reported during or after steroid therapy. 4. Discussion Several studies, including our results, demonstrated that an excessive host-cellular response, especially IL-18, plays a critical role in the development of serious pulmonary manifestations [12e15], and serum levels of IL-18 were significantly correlated
with those of LDH [14,15]. Because the measurement of serum IL-18 levels cannot be always performed, LDH instead of IL-18 was thought to be useful as a predictor of refractory or severe M. pneumoniae pneumonia in the daily clinical setting. Therefore, in this study we analyzed serum LDH levels consecutively in order to set a standard for the initiation of steroid therapy in M. pneumoniae pneumonia. In our preliminary study among pediatric patients, we demonstrated that steroid administration would be required when serum LDH levels are 410 IU/L [15]. However, the sample size was
Fig. 4. Changes in serum LDH levels in 11 patients with refractory M. pneumoniae pneumonia at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
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Fig. 5. Changes in serum LDH levels in 19 patients with severe M. pneumoniae pneumonia who required mechanical ventilation at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
very small (n ¼ 5) to determine the appropriate criterion for the initiation of steroid therapy. In this study, our results suggested that the serum LDH cut-off level was 364 IU/L at initiation of steroid therapy and 302 IU/L at 1e3 days before the initiation of steroid therapy in patients with refractory and severe M. pneumoniae pneumonia. To prevent the progression of pneumonia severity, serum LDH levels of 302e364 IU/L seemed to be an appropriate criterion for the initiation of steroid therapy. Our results also demonstrated that steroids combined with appropriate antibiotic therapy reduced serum LDH levels in the
refractory and severe groups to the same level as in the control group (Figs. 3e6). Thus, serum LDH levels may be a useful marker for the evaluation of therapeutic efficacy in refractory and severe M. pneumoniae pneumonia. Histopathologically, M. pneumoniae pneumonia is characterized by acute cellular bronchiolitis and by peribronchial and perivascular interstitial opacities. These histopathological findings closely reflect chest HRCT findings, which indicate that the combination of bronchial wall thickening and centrilobular nodules [25]. However, at the progressed stage refractory and severe
Fig. 6. Changes in serum LDH levels in 11 patients with severe M. pneumoniae pneumonia who did not required mechanical ventilation at five points before and after antibiotic and steroid therapy. Bars indicate range and squares indicate median value ± interquartile range.
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Table 2 Serum LDH cut-off value, sensitivity, specificity, and area under the receiver operating characteristic curve for the initiation of steroid therapy in patients with refractory and severe M. pneumoniae pneumonia. Variables At the initiation of steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients At 1e3 days before steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients At 4e7 days before steroid therapy Refractory group Severe group with mechanical ventilation Severe group without mechanical ventilation All refractory and severe patients
LDH cut-off value (IU/L)
Sensitivity (%)
Specificity (%)
Area under the ROC curve
395 367 364 364
100 89.5 90.9 92.7
100 99.1 99.1 99.1
1 0.992 0.989 0.993
302 363 312 302
100 84.2 90.9 92.7
92.6 99.1 92.6 92.6
0.984 0.983 0.981 0.983
258 264 243 243
63.3 63.2 72.7 70.7
84.3 87.0 79.6 79.6
0.769 0.808 0.808 0.804
LDH, lactate dehydrogenase; ROC, receiver operating characteristic.
patients showed bilateral diffuse infiltrates and did not show the radiographic features of M. pneumoniae pneumonia that was observed in the non-progressed stage (control group). Because of the non-specific radiographic findings, chest HRCT findings thought to be not useful parameter to determine the point to initiate steroid therapy in refractory or severe M. pneumoniae pneumonia. Before initiating steroid therapy in CAP patients, we must be define or suspect M. pneumoniae infection, because there are no evidence to support the well tolerated use of systemic steroids in CAP due to other pathogens [26e28]. Among CAP pathogens, we have demonstrated that the presumptive diagnosis of M. pneumoniae pneumonia is relatively easy using the combination of three rapid and simple diagnostic methods: the JRS scoring system, chest computed tomography, and ImmunoCard Mycoplasma [20e22]. In addition, two commercially available point-ofcare test kits for detection of the M. pneumoniae antigen, Ribotest
Community-acquired pneumonia patients with: 1. met all parameters out of six of the JRS scoring system or 2. positive for M. pneumoniae rapid antigen tests or 3. met four or more parameters out of six of the JRS scoring system + chest CT findings
Initiation of anti-M. pneumoniae antibiotics
Deterioration of clinical and radiographic findings
Check for serum LDH level consecutively
Serum LDH level: 302-363 IU/L
Serum LDH level: ≥ 364 IU/L
Consider to initiation of steroid therapy
Initiation of steroid therapy
Fig. 7. A stepwise algorithm to correlate diagnosis, serum markers, and initiation of steroid therapy in M. pneumoniae pneumonia.
Mycoplasma (Asahikasei Pharma Co., Tokyo, Japan) and PrimeCheck Mycoplasma Antigen (Alfresa Pharma Co., Osaka, Japan) have been available since 2013 in Japan [29]. Using the combination of these five rapid and simple methods, the diagnostic sensitivity and specificity of M. pneumoniae pneumonia have increased. A stepwise algorithm to correlate diagnosis, serum markers, and initiation of steroid therapy is summarized in Fig. 7. M. pneumoniae showing resistance to macrolides was isolated from patients with CAP in 2000, and macrolide resistance has become widespread in Japan [22,23]. In our previous studies, no significant differences in pneumonia severity were observed between macrolide-resistant patients and macrolide-sensitive patients [22,23]. In this study, the prevalence of macrolide-resistant strain was almost identical among control and severe groups. Thus, macrolide-resistant strain seems to be not related with pneumonia severity. Pulmonary injury associated with refractory or severe M. pneumoniae pneumonia thought to be due to an excessive, activated cell-mediated immune response to pathogens rather than to direct microbial damage [1,13]. If refractory or severe M. pneumoniae pneumonia is an immune mediated disease, immune suppressive therapy is a rational approach. In our study, corticosteroid combined with antibiotics therapy demonstrated the satisfactory clinical effects and reduced serum IL-18 and LDH levels in patients with refractory or severe M. pneumoniae pneumonia [3,15]. The use of corticosteroids combined with antibiotics therapy for decreasing the inflammatory response with dramatic beneficial effect has also been demonstrated in several studies [4e10,14,16,17]. Thus, high level of serum IL-18 and LDH levels in refractory or severe group may be due to host immune response rather than to virulence of M. pneumoniae. However, no available data for explanation of mechanism in which linked the raised serum IL-18 and LDH levels in refractory or severe group. Further study is needed. Our study had several limitations. Since present study was not case-control study, our study lacks the data of explaining the benefits of using steroid for the patients with refractory or severe M. pneumoniae pneumonia who had high value of serum LDH. Further prospective studies are needed to confirm the beneficial effect of steroids in patients with high value of serum LDH. Second, we could not demonstrate the presence of macrolide-resistant mutations in all patients. Thus, in this study, we selected minocycline or quinolones as antibiotics, because these agents are clinically and bacteriologically more effective than macrolides [22,23]. The change in serum LDH levels in patients with M. pneumoniae
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pneumonia in both macrolide-resistant and macrolide-sensitive strains who were treated with macrolides should be investigated. Third, it has been suggested that elevated serum ferritin and urine b2-microglobulin levels, which suggest complications of hypercytokinemic conditions, may be associated with the severity of pneumonia [6]. In addition, the strong correlation between serum cortisol and adrenocorticotropic hormone levels and pneumonia severity has been reported [30]. In this study, however, we did not evaluate these markers because the results cannot be obtained promptly. Finally, we enrolled only CAP cases who met all parameters out of six of the JRS scoring system because of the selection of definitive cases with M. pneumoniae pneumonia exactly. Thus, we missed many cases with M. pneumoniae pneumonia during the study period. The number of patients including refractory or severe cases reflects selection bias. In conclusion, our results suggest that the serum LDH level can be a useful parameter to determine the point to initiate steroid therapy in refractory or severe M. pneumoniae pneumonia. A serum LDH level of 302e364 IU/L seemed to be an appropriate criterion for the initiation of steroid therapy. In addition, serum LDH level seems to be a useful marker for the evaluation of therapeutic efficacy. 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, 19402M, 20-4030). Appendix This study was conducted by following institutions; Kato T. (Asahigaoka Hospital); Yamada H. (Takahashi Central Hospital); Kawasaki F. (Okayama Saidaiji Hospital); Tanaka H. (Saijo Aijukai Hospital); Okimoto N. (Satou Memorial Hospital); Masuda T. (Tamano Central Hospital); Nakatani S. (Nakatani Geka Hospital); Maruyama T. (Mie National Hospital); Nakahama C. (Nakahama Clinic); Yoneyama H. and Nakamura J. (Kasaoka Daiichi Hospital); Kurihara T. (Kawasaki Hospital); Kawanishi M. (Kaneda Hospital); Kimura M. (Kimura Clinic); Hara H. and Matsushima T. (Kurashiki Daiichi Hospital); Yano T. (Kurashiki Memorial Hospital); Yagi S. (Heisei Hospital); Ikeda M. (Kojima Central Hospital); Mouri K. (Tamashima Central Hospital); Kato T. (Nakashima Hospital); Sugimura S. (Okayama Kyoritsu General Hospital); Doi M. and Kuwabara M. (Kenritsu Hiroshima Hospital); Hashiguchi K. (Nagasaki Genbaku Hospital); and Nagatomo Y. and Okayama A. (University of Miyazaki). References [1] Waites KB, Talkington DF. Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 2004;17:697e728. [2] National Institute of Health. Infectious Disease Surveillance Center. Mycoplasma pneumoniae pneumonia. http://idsc.nih.go.jp/idwr/kanja/ weeklygraph/18myco.html. [3] 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. [4] 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.
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