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Diagnostic value of quantitative MP-IgG for Mycoplasma pneumoniae pneumonia in adults
Journal Pre-proofs Diagnostic value of quantitative MP-IgG for Mycoplasma pneumoniae pneumonia in adults Lina Wu, Maosheng Ye, Xiaosong Qin, Yong Liu, Zhe Lv, Rui Zheng PII: DOI: Reference:
S0009-8981(20)30012-7 https://doi.org/10.1016/j.cca.2020.01.004 CCA 15984
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Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
14 March 2019 7 January 2020 7 January 2020
Please cite this article as: L. Wu, M. Ye, X. Qin, Y. Liu, Z. Lv, R. Zheng, Diagnostic value of quantitative MPIgG for Mycoplasma pneumoniae pneumonia in adults, Clinica Chimica Acta (2020), doi: https://doi.org/ 10.1016/j.cca.2020.01.004
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Diagnostic value of quantitative MP-IgG for Mycoplasma pneumoniae pneumonia in adults Lina Wu1, Maosheng Ye2, Xiaosong Qin1, Yong Liu1, Zhe Lv1, Rui Zheng2* 1 Department of Laboratory Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China, 2 Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China *Corresponding author at: Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China
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Abstract The passive particle agglutination (PA) test1, once widely used for Mycoplasma pneumoniae (M. pneumoniae) antibody detection, has gradually been replaced by quantitative enzyme-linked immunosorbent assays (ELISA). However, the lack of diagnostic criteria for quantitative ELISA M. pneumoniae-IgG(MP-IgG) and the low positive rates of ELISA M. pneumoniae-IgM (MP-IgM) limit the diagnostic value of ELISA for M. pneumoniae infection in adults. Here, the diagnostic value of quantitative ELISA MP-IgG was evaluated in adults with Mycoplasma pneumoniae pneumonia (MPP). The serum M. pneumoniae antibodies were detected in 162 patients with MPP, 228 patients with community-acquired pneumonia (CAP) with non-Mycoplasma pneumoniae(NMP), and 162 healthy controls by ELISA, using the PA results as the reference standards. For the MP-IgM-/IgG+ subgroup, a single serum MP-IgG level of ≥92.67 RU/mL can be used as a reference criterion for the diagnosis of acute M. pneumoniae infection. At admission, for patients with CAP, the sensitivity and specificity of ELISA MP-IgM positivity for MPP were 18.51% and 99.56%, respectively. MP-IgM positivity combined with MP-IgG ≥92.67 RU/mL increased the sensitivity to 40.12% and decreased the specificity to 94.29%. For paired serum samples obtained within seven days, an ELISA MP-IgG concentration change of ≥ 1.48-fold and MP-IgG ≥ 92.67 RU/mL on day 7 were used as the diagnostic criteria for M. pneumoniae infection. Accordingly, the combination of qualitative MP-IgM detection and quantitative MP-IgG detection by ELISA is
Abbreviations: PA , passive particle agglutination ; M. pneumoniae , Mycoplasma pneumoniae ; ELISA , enzyme-linked immunosorbent assays ; MPP , Mycoplasma pneumoniae pneumonia ; CAP , community-acquired pneumonia ; NMP, non-Mycoplasma pneumoniae; MP-IgG: M. pneumoniae-IgG; MP-IgM: M. pneumoniae-IgM Corresponding author at: Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China E-mail address: [email protected] 2
valuable for acute MPP diagnosis in adults.
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Keywords: Mycoplasma pneumoniae; passive particle agglutination test; enzyme-linked immunosorbent assay; IgG; IgM
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1. Introduction Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen leading to community-acquired pneumonia (CAP) in adults [1-4]. Owing to its lack of a cell wall, M. pneumoniae is intrinsically resistant to cell wall-targeting antibacterial drugs, such as β-lactams [5], and is only sensitive to fluoroquinolones, macrolides, and tetracyclines. To avoid treatment delay, an increasing number of patients with suspected M. pneumoniae infection are treated with macrolide antibiotics based on clinical symptoms and imaging findings during the early stage, despite the difficulty in differentiating Mycoplasma pneumoniae pneumonia (MPP) from CAP caused by other pathogens using this approach [6]. In addition, after the infections are cured, M. pneumoniae might remain in the respiratory tract mucosa [7-9]. As macrolides are widely used in clinical practice, the prevalence of macrolide resistance has also increased worldwide [10-12]. Compared with that of patients with macrolide-sensitive M. pneumoniae infection, patients with macrolide-resistant M. pneumoniae infection experience prolonged persistent cough, fever, and antibiotic treatment [13-18]. Therefore, it is essential to select the optimal laboratory tests and establish effective diagnostic criteria for obtaining a definitive diagnosis, initiating timely and effective treatment, and reducing the high rate of drug-resistance due to the misuse of antibiotics for patients infected with M. pneumoniae. A rapid and reliable serologic test for M. pneumoniae antibodies can provide strong evidence for diagnosis. The passive particle agglutination (PA) test is conventionally used to detect M. pneumoniae-specific antibodies in the serum, and its diagnostic value for M. pneumoniae infection has been demonstrated in clinical settings [1,19,20,21]. However, PA is prone to subjectivity with respect to the manual
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procedures and interpretation of results. In addition, PA cannot differentiate between IgM and IgG antibodies. As is known to all, MP-IgM appears at the 1st week, and achieves the peak at 3 weeks after M. pneumoniae infection. MP-IgM positive is of important reference significance for the early or acute diagnosis of M. pneumoniae infection. Therefore, this test may be gradually replaced by quantitative enzyme-linked immunosorbent assays (ELISA). Compared with the PA test, the ELISA can be used to detect M. pneumoniae-IgM (MP-IgM) and M. pneumoniae-IgG (MP-IgG) antibodies, respectively. In addition, the use of an automated enzyme immunoassay analyzer enables easy MP-IgM and MP-IgG detection and the objective interpretation of results. This has improved the accuracy of test results and has almost overcome the limitations of PA. It has been reported that some adult patients with repeated M. pneumoniae infection have sustained MP-IgM negative [22]. In this study, the ratio of MP-IgM-/IgG+ subgroup was the highest in MPP, CAP with NMP, control groups; and the concentration of MP-IgG in the MPP group was significantly higher than that in the CAP with NMP and control groups at the same time during hospitalization. This indicates that the quantitative ELISA MP-IgG results will be of great value for the diagnosis of M. pneumoniae infection. However, as the results of ELISA are not expressed in titers but in S/CO or concentrations, the previous diagnostic criterion for the detection of M. pneumoniae infection by PA (a 4-fold or greater change in the titer between paired acute- and convalescent-phase serum samples) is not applicable [23,24]. Therefore, there is an urgent need to establish diagnostic criteria for the ELISA for M. pneumoniae infection. Lee et al. [25] have
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suggested that for patients with severe conditions, especially for hospitalized patients, a titer change of 4-fold or greater within one week is helpful for the early diagnosis of M. pneumoniae infection. In the present study, an increase in paired serum titers of ≥4-fold within seven days or an initial serum titer of ≥1:640 with an increase of ≤2-fold by the PA test were used as the inclusion criterion for the MPP group of patients with CAP. We evaluated the diagnostic value and criteria of qualitative ELISA MP-IgM and quantitative ELISA MP-IgG for adults with MPP.
2. Materials and methods 2.1. Subjects Between November 2016 and June 2018, 390 patients with confirmed CAP in the Department of Pulmonary and Critical Care Medicine of Shengjing Hospital of China Medical University were included in the patient group. A total of 162 healthy individuals who visited the physical examination center and had no respiratory tract infection within the last six months with normal chest CT, white blood cell (WBC) count, and C-reactive protein (CRP) level were included in the control group. Mycoplasma pneumoniae infection was diagnosed based on the following internationally recognized criterion: a 4-fold or greater change in the serum M. pneumoniae-specific antibody titers from acute- and convalescent-phase paired serum samples [23, 24]. Patients with CAP (n = 390) were divided into the MPP group and the CAP with NMP group based on the changes in serum M. pneumoniae antibody titers detected by PA between the time of admission and seven days after admission. The MPP group was further divided into three subgroups according to the changes in M. pneumoniae antibodies detected by PA. Subgroup 1 included 20 patients with 7
seroconversion (i.e., negative for M. pneumoniae antibodies at admission, with a titer of ≥ 1:160 seven days after admission). Subgroup 2 included 138 patients whose M. pneumoniae antibody titers increased to ≥ 4-fold within seven days. Subgroup 3 included four patients with M. pneumoniae antibody titers of ≥1:640 at admission, with a change in M. pneumoniae antibody titers of ≤ 2-fold within seven days . Patients with CAP who did not meet the above criteria were assigned to the CAP with NMP group. Clinical characteristics, including age, gender, and symptoms of patients in the MPP, CAP with NMP, and healthy control groups are shown in Table 1; Age distribution of each group is detailed in Table S1. Fasting venous blood (2 mL) was drawn from patients in the MPP and CAP with NMP groups at admission and seven days after admission. The blood samples were centrifuged at 1200g for10 minutes, and the serum was separated and stored in a -70°C freezer. The leftover serum from healthy individuals after physical examination was collected and stored in a -70°C freezer. All samples were collected and tested centrally in one laboratory. This study was approved by the ethics committee (approval number: 2018PS429K) of Shengjing Hospital of China Medical University.
2.2. Methods PA was used to detect M. pneumoniae antibodies. The ELISA was used to detect MP-IgM and MP-IgG antibodies. Reagents for PA (SERODIA®-MYCOII) and the ELISA were purchased from FUJIREBIO Co., Ltd. (Tokyo, Japan) and EUROIMMUN Medical Laboratory Diagnostics (Lubeck, Germany), respectively. PA was used to detect a mixture of M. pneumoniae antibodies and the results are expressed as follows: negative (titer: <1:40) and positive (titers: 1:40, 1:80, 1:160, 1:320, 1:640, and ≥1:1280). In order to ensure the accuracy of M. pneumoniae antibody with PA, the whole operation was carried out by training and assessing 8
technicians according to the standard operating procedure of M. pneumoniae antibody with PA. Each test result will be interpreted separately by two technicians. If consistent, the result will be reported to the clinic. But if not, the result will be made a check by the competent technician or re-test if necessary. The ELISA was used to detect MP-IgM and MP-IgG antibodies by TECAN automated enzyme immunoassay analyzer . According to the manufacturer’s protocol of the ELISA (EUROIMMUN Medical Laboratory Diagnostics, Lubeck, Germany) , the results of ELISA MP-IgM based on S/CO were interpreted as follows: negative if <0.8, equivocal if 0.8–1.1, and positive if ≥1.1. The results of ELISA MP-IgG were interpreted as follows: negative if <16 RU/mL, equivocal if 16–22 RU/mL, and positive if ≥22 RU/mL. For both ELISA MP-IgM and MP-IgG, samples with results within the equivocal range were tested at least twice again. If the coefficient of variation of multiple test results is less than 10% and not more than 1.1 times of the precision within-run of the corresponding concentration provided by the kit instruction, the mean of the multiple test results is taken to judge the negative or positive results. If the mean is still in the equivocal range or positive, it is counted as positive; otherwise, negative.
2.3. Statistical analysis Qualitative data are expressed as percents and were analyzed using the χ2 test. Quantitative data that followed a normal distribution were expressed as mean ± —
standard deviation (X ± S) and were analyzed using the t-test or analysis of variance. Quantitative data that did not follow a normal distribution were expressed as median (interquartile range) and were analyzed using the Kruskal–Wallis H test. Correlation analyses were conducted using linear regression. All statistical analyses were performed using SPSS 20.0. Differences with P < 0.05 were considered statistically 9
significant.
3. Results 3.1. At admission, both the positive rate and titer in MPP group as determined by the PA test were significantly higher than those in the CAP with NMP group and the control group Based on a qualitative analysis using 1:40 as the cut-off value for M. pneumoniae infection, the positive detection rate for the MPP, CAP with NMP, and control groups was 87.65%, 44.74%, and 44.44%, respectively, and the positive rate in the MPP group was significantly higher than that in the CAP with NMP and control groups (Table 2, P ＜0.001). For a titer of ≥1:160, the positive rate in the MPP group (38.88%) was significantly higher than that in the CAP with NMP(6.14%) and control groups (7.41%) (Table 2, P＜0.001). At admission, the median titer in the MPP group (1:80) was significantly higher than that in the CAP with NMP (<1:40) and control groups (<1:40) (Figure 1, P＜0.001), and there was no statistical difference between the CAP with NMP and control group (Figure 1). 3.2. The proportion of ELISA MP-IgG single positive patients was the highest in the MPP, CAP with NMP groups during hospitalization and control group According to the qualitative results for MP-IgM and MP-IgG obtained by the ELISA at admission, patients in the three groups could be divided into the following four subgroups: IgM and IgG positive (MP-IgM+/IgG+), IgM single positive (MP-IgM+/IgG-), IgG single positive (MP-IgM-/IgG+), and IgM and IgG negative (MP-IgM-/IgG-). In the MPP group, the CAP with NMP group during hospitalization, and the control group, the MP-IgM-/IgG+ subgroup was the most frequent among the four subgroups (Table 3, P＜0.001). 3.3. For the MP-IgM-/IgG+ subgroup, the level of MP-IgG in the MPP group was 10
significantly higher than that in CAP with NMP and control groups At admission, the quantitative results for the MP-IgG in MP-IgM-/IgG+ subgroup showed that the median level of MP-IgG in the MPP group [ n = 101, 59.74 (33.01-96.96) RU/mL] was significantly higher than that in the CAP with NMP [ n = 147, 39.69 (23.34-61.43) RU/mL, P < 0.001] and control groups [n = 131, 50.00 (28.35-71.56) RU/mL, P = 0.041] (Figure 2A). At seven days after admission, the median level of MP-IgG in the MPP group [n = 110, 156.84 (138.48-187.77) RU/mL] was significantly higher than that in the CAP with NMP [n = 138, 45.07 (26.48-66.66) RU/mL, P < 0.001] and control groups [n = 131, 50.00 (28.35-71.56) RU/mL, P < 0.001] (Figure 2B). 3.4. For MP-IgG single positive patients, the concentration of ELISA MP-IgG was 92.67RU/mL, which may be used as a reference criterion to the diagnosis of M. pneumoniae infection Similar to the titer change in paired serum samples obtained by the PA test for the MPP group, the ELISA also showed seroconversion and increase in antibody concentrations (Table 4). To explore the correlation between the concentration of MP-IgG obtained by the ELISA and the titers obtained by the PA test, we evaluated patients who were MP-IgM-negative during hospitalization in the MPP group (n = 110, specimens = 220, Table 4), CAP with NMP group (n = 227, specimens = 454, Table 3), and MP-IgM-negative controls (n = 162, specimens = 162, Table 3). The ELISA MP-IgG levels were highly correlated with the PA titers (Y = 24.85 + 23.74X, F = 1149.91, where Y and X represent the concentration of MP-IgG by the ELISA and PA titer, respectively, and the titers obtained by the PA test (from <1:40 to 1:10240) were replaced with a value of 0 to 9, P < 0.001) (Figure 3). Corresponding to a titer of 1:160 using the PA test, the 95% confidence interval of MP-IgG levels determined by 11
the ELISA was 92.67 - 99.47 RU/mL. 3.5. For MP-IgM-/IgG+ subgroup, an ELISA MP-IgG concentration change of ≥ 1.48-fold can be used as an exact criterion to the diagnosis of M. pneumoniae infection To explore the MP-IgG concentration change in the acute phase of M. pneumoniae infection by the ELISA, we evaluated patients with seroconversion or increased antibody titers (≧4) using the PA test and solitary ELISA MP-IgG-positive during hospitalization in the MPP group (n = 92, Table 4). The median and one-sided 95% confidence interval of MP-IgG increase in paired serum samples by the ELISA were 2.45 (1.69-4.34) (Figure 4) and ≧1.48, respectively. Furthermore, for patients identified as solitary IgG-positive during hospitalization in the CAP with NMP group (n = 137), the median and one-sided 95% confidence interval for increase in ELISA MP-IgG were 1.01 (0.88-1.12) (Figure 4) and ≤1.53, respectively .
4. Discussion In this study, PA was used as the reference standard to investigate the diagnostic value and threshold criteria for ELISA MP-IgM and MP-IgG in adults with MPP. For MPP diagnosis in adults with CAP, at admission and on day 7 after admission, the diagnostic sensitivity of ELISA MP-IgM positivity were 18.51% and 32.10% (Table S5), respectively. This indicates that ELISA MP-IgM positivity alone is insufficient for the diagnosis of M. pneumoniae infection in adults. The subgroup with single positive results for ELISA MP-IgG accounted for the highest proportion of patients in the MPP, CAP with NMP, and control groups; at admission and on day 7 after admission, the concentration of MP-IgG in the MPP group was significantly higher than that in the CAP with NMP and control groups at the same time points. This suggests that the quantitative MP-IgG results obtained by the ELISA are of great 12
value for the diagnosis of M. pneumoniae infection. Further investigations showed that in the MP-IgG-single positive subgroup, a single serum ELISA MP-IgG level of ≥ 92.67 RU/mL can be used as a reference criterion for the diagnosis of M. pneumoniae infection, whereas a change in the concentration of MP-IgG by ELISA within seven days of ≥1.48-fold in the paired serum samples and MP-IgG ≥92.67 RU/mL on day 7 after admission can be used as the diagnostic criteria for acute M. pneumoniae infection. Although a positive MP-IgM result for a single serum sample can be used as a diagnostic reference for M. pneumoniae infection [23], it does not provide a sufficient basis for the diagnosis of M. pneumoniae infection in adults. The positive detection rate of MP-IgM in the MPP group in the present study was consistent with the results of Martinez et al., who showed that the diagnostic sensitivity of MP-IgM for acute M. pneumoniae infection in adults was 33.3% [26]. It has been reported that the positive detection rate of MP-IgM is related to age. Chamberlain et al. performed a complement fixation test and found that the positive rate of MP-IgM in patients with MPP under 20 years of age was approximately 78%, whereas that in adult patients was only 34% [27]. Moule et al. [28] performed ELISA (μ-capture) and found that approximately 91% of patients under the age of 20 were positive for MP-IgM, whereas the positive rate in adult patients was approximately 50%. Initial M. pneumoniae infection can lead to the production of memory B cells and T cells, which are rapidly activated upon reinfection. This is manifested in some patients as a rise in MP-IgM after 2-3 days of infection, followed by a peak in the MP-IgM level within a short period and a rapid increase in IgG thereafter; other patients may show an immediate rise in the IgG level. Therefore, we speculate that the differences in the positive detection rate of MP-IgM among age groups following M. pneumoniae
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infection may be related to differences in the proportion of patients re-infected with M. pneumoniae. Luciani L et al have defined residual antibodies as low or intermediate rate of IgG against Coxialla burnetii that can persist for several months or even years after primary infection, without active infection.Therefore, IgG is a well-established indicator of previous infection [29]. In this study, the high positive rate of MP-IgG in the MPP, CAP with NMP groups during hospitalization and control group suggest a high proportion of adults with M. pneumoniae reinfection. Specifically, in the MPP group, 101 and 110 patients were MP-IgG single positive at admission and seven days after admission, accounting for 62.35% and 67.90%, respectively; 7 days after admission, 9 patients with MP-IgG single positive at admission converted to MP-IgM+/MP-IgG+, and 18 patients with MP-IgM-/MP-IgGat admission converted to MP-IgG single positive. In the CAP with NMP group, 147 and 138 patients were MP-IgG single positive at admission and seven days after admission, accounting for 64.47% and 60.53%, respectively; 7 days after admission, 10 patients with MP-IgG single positive at admission converted to MP-IgM-/MP-IgG-, and 1 patients with MP-IgM+/MP-IgG+ at admission converted to MP-IgG single positive. In view of the high proportion of adults with M. pneumoniae reinfections and assuming that the time from symptom onset to hospital visit is generally longer than one week, the scientific use of quantitative MP-IgG results as a reference for the diagnosis of M. pneumoniae infection may compensate for the diagnostic limitations of MP-IgM, to some extent. Yoshimasu et al. [30] have reported that a high titer of anti-Helicobacter pylori IgG antibodies may be an indicator for upper gastrointestinal endoscopy and eradication therapy in patients with acute or chronic urticaria. Sonmez et al. [31] have proposed that the anti-PT IgG antibody levels of ≥100 EU/mL should
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be considered as an indicator of acute or recent infection with Bordetella pertussis. IgG and IgM are the most important antibodies used in serologic testing for M. pneumoniae infection. Owing to the potential lack of an IgM response in adults re-infected with M. pneumoniae, the IgG level is particularly important for the diagnosis of M. pneumoniae reinfection [32]. In the present study, in the subgroup of patients positive for MP-IgG only, both at admission and seven days after admission, the median MP-IgG concentration in the MPP group was significantly higher than that in the CAP with NMP and control groups. And in the MPP group, the MP-IgG concentration on seven days after admission was significantly higher than that at admission (P ＜ 0.001, Data in Figure 2A and Figure 2B). However, there was no significant difference in MP-IgG concentration between the two time points in the CAP with NMP group (P = 0.318, Data in Figure 2A and Figure 2B) . This suggests that the concentration of MP-IgG can be used as a reference for the diagnosis of acute M. pneumoniae infection. There is a consensus in available literature that a single serum M. pneumoniae antibody titer of ≥ 1:160 detected by PA should be used as a reference for the diagnosis of recent M. pneumoniae infection [22]. For patients who were MP-IgM-negative during hospitalization in the MPP, CAP with NMP, and the control groups, we found that the level of MP-IgG determined by the ELISA was correlated with the PA titers. Corresponding to a titer of 1:160 for the PA test, the 95% confidence interval of MP-IgG by the ELISA was 92.67 - 99.47 RU/mL. The combination of MP-IgM positivity and MP-IgG ≥ 92.67 RU/mL as a reference for the diagnosis of acute M. pneumoniae infection can significantly improve the diagnostic sensitivity and positive predictive value for M. pneumoniae infection. When the two thresholds were combined, the sensitivity and specificity for the diagnosis of MPP in patients with CAP at admission were 40.12% and 94.29%
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(Table S6), respectively, which were slightly higher than those for a titer of 1:160 by PA (i.e., 38.88% and 93.86%, Table S7). When compared with the positivity for MP-IgM alone (18.51% and 99.56%), the diagnostic sensitivity improved significantly, while the diagnostic specificity decreased. According to the health industry standard of the People’s Republic of China, WS/T 494-2017 “Guideline for performance characteristics of immunological qualitative test”, the diagnostic tests are used to detect the presence of clinically suspected analytes, such as infectious pathogen antigens or related antibodies. If the infective pathogen antigen or related antibody to be tested is important for treatment and prognosis, the diagnostic test should be sensitive enough on the premise of ensuring certain clinical specificity (≥ 90%) [33]. As a diagnostic test for acute or recent infection of M. pneumoniae, the results of serum M. pneumoniae antibody are very important for scientific selection of treatment options and reduction of M. pneumoniae with macrolides resistant. The serological test of M. pneumoniae antibody should be sensitive enough on the premise of ensuring certain clinical specificity ( ≥ 90%). Therefore, The combination of MP-IgM positivity and MP-IgG ≥ 92.67 RU/mL is more suitable as a reference standard for acute or recent M. pneumoniae infection. Clinicians prefer the use of positive predictive value (PPV) and negative predictive value (NPV) over diagnostic sensitivity and specificity. Based on the sensitivity and specificity of the ELISA and PA as well as the assumption that the incidence of MPP in patients with CAP was 30%, the combination of MP-IgM positivity and MP-IgG ≥92.67 RU/mL would correspond to a PPV and NPV of 75.07% and 78.61% (Table S8), respectively, both of which were higher than those for PA (PPV: 73.07%, NPV: 78.18%, Table S9). In addition to the high proportion of patients positive for MP-IgG only at admission, this study also included a certain proportion of patients who were negative
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for both IgM and IgG. To avoid missed diagnosis, we collected paired serum samples from patients during hospitalization and found that 31 patients in the MPP group were negative for both IgM and IgG at admission. At seven days after admission, 13 patients presented positive for both IgM and IgG (six patients with MP-IgG < 92.67 RU/mL), and 18 patients had significant seroconversion for IgG only (ten patients with MP-IgG < 92.67 RU/mL). The occurrence of seroconversion supports the diagnosis of acute M. pneumoniae infection. However, for the subgroup of patients positive for MP-IgG only during hospitalization, the proper use of a concentration change in the paired serum samples for the diagnosis of M. pneumoniae infection is a major concern for clinicians.As mentioned above, a change in titers of ≥ 4-fold between the acute and convalescent phases is a definitive criterion for the diagnosis of M. pneumoniae infection [23,24]; however, this criterion is based on qualitative and visual assessments of the PA and ELISA results. This criterion enables a preliminarily determination of the change in antibody concentration by gradually diluting the sample, under the premise that the antibody concentration itself cannot be accurately detected. Owing to the development of automated enzyme immunoassay analyzers in the 1990s, the current ELISA kits no longer use titers to determine results, but rather directly calculate the S/CO values or antibody concentrations in the serum using microplate readers to obtain semiquantitative and quantitative results. Therefore, the above diagnostic criterion no longer applies to the current ELISA kits. Loens et al. [34] suggested that the clinical significance of serologic tests for IgG should be determined by studies on patients with documented infection and detailed information on the time lapse between disease onset and the collection of serum specimens. Therefore, to investigate the changes in serum MP-IgG concentrations detected by the ELISA in the acute phase of M. pneumoniae infection, we enrolled 92
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patients in the MPP group whose paired serum samples collected during hospitalization showed seroconversion or an antibody titer increase of ≥4-fold as detected by the PA test, with positive results for ELISA MP-IgG only. In a comparative analysis, the one-sided 95% confidence interval for concentration changes detected in paired serum samples by ELISA MP-IgG in the MPP and CAP with NMP groups was ≥1.48- and ≤1.53-fold, respectively. This suggests that a concentration change in paired serum MP-IgG between 1.48 and 1.53 is a gray area for diagnosis. Further analysis of the 17 patients in CAP with NMP group whose change in MP-IgG concentration was ≥1.48 revealed that the MP-IgG concentration at admission was between 16.00 and 30.00 RU/mL and the MP-IgG concentration increased seven days after admission to between 24.00 and 60.00RU/mL. These concentrations were lower than 92.67 RU/mL at both time points. Conversely, the MP-IgG concentration in all 92 patients in the MPP group was > 92.67 RU/mL at seven days after admission. Further investigations of patients in the MPP group whose change in the paired sera concentration within seven days was <1.48 showed that the MP-IgG concentration was approximately 200 RU/mL in all patients during hospitalization. The upper limit of quantification for the ELISA MP-IgG kit used in this study was 200 RU/mL; the validity of these results as diagnostic criteria for M. pneumoniae infection could not been confirmed owing to the limited number of cases in this study. Therefore, the following can be used as diagnostic criteria for acute M. pneumoniae infection in adults with positive ELISA MP-IgG only: ≥1.48-fold difference in the MP-IgG concentration within seven days and an MP-IgG concentration in the second serum sample of ≥ 92.67 RU/mL. In summary, the combination of qualitative MP-IgM detection and quantitative MP-IgG detection by ELISA is of significant importance for the diagnosis of MPP in
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adults. To date, quantitative ELISA MP-IgG has not been used as a reference standard and diagnostic criterion for the diagnosis of MPP in adults. Nevertheless, M. pneumoniae antigens from different manufacturers have different origins and traceability standards. Therefore, a limitation of the study is that the results are only applicable to the ELISA kits with the same traceability as that of the kit used in this study. Acknowledgments We would like to express our gratitude to all of the subjects who participated in this study. Potential conflict of interest We declare that no competing interests exist. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions Conceived and designed the experiments: Lina Wu, Rui Zheng. Performed the experiments: Lina Wu, Maosheng Ye, Zhe Lv. Analyzed the data: Lina Wu, Xiaosong Qin. Contributed reagents/materials/analysis tools: Lina Wu, Yong Liu. Wrote the paper: Lina Wu, Rui Zheng.
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Figure Legends Figure 1. The titers of M. pneumoniae antibodies with PA in the MPP, CAP with NMP and control groups at admission. M. pneumoniae: Mycoplasma pneumoniae, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, PA: passive particle agglutination. Data was expressed as median, and analyzed using the Kruskal–Wallis H test. P < 0.05 was considered statistically significant. *P<0.001,
MPP group vs. CAP with NMP group；#P<0.001, MPP group vs. control group
Figure 2. The concentration of ELISA MP-IgG in the MP-IgM-/IgG+ subgroup in the MPP, CAP with NMP groups during hospitalization and control group. MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, ELISA: enzyme-linked immunosorbent assays, MP-IgM: Mycoplasma 23
pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive. Data was expressed as median (interquartile range), and analyzed using the Kruskal–Wallis H test. P < 0.05 was considered statistically significant. A. at admission:*P<0.001, MPP group [59.74 (33.01-96.96) RU/mL] vs. CAP with NMP group [39.69 (23.34-61.43) RU/mL];
#P=0.041,
MPP group [59.74 (33.01-96.96) RU/mL] vs. control group [50.00
(28.35-71.56) RU/mL]. B. 7 days after admission: *P<0.001, MPP group [156.84 (138.48-187.77) RU/mL] vs. CAP with NMP group [45.07 (26.48-66.66) RU/mL]; #P<0.001, MPP group [156.84 (138.48-187.77) RU/mL] vs. control group [50.00 (28.35-71.56) RU/mL].
Figure 3. The correlation between the concentration of MP-IgG in ELISA and the titer of PA. M. pneumoniae: Mycoplasma pneumoniae, PA: passive particle agglutination, ELISA: enzyme-linked immunosorbent assays, MP-IgG: Mycoplasma pneumoniae-IgG. The titers of PA from ＜ 1:40 to 1:10240 were replaced by 0 to 9 respectively, and the equation of regression was Y=24.85+23.74X, F=1149.91, P ＜0.001.
Figure 4. The increasing times of MP-IgG with ELISA within 7 days in MPP and CAP with NMP groups. ELISA: enzyme-linked immunosorbent assays, MP-IgG: Mycoplasma pneumoniae-IgG, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group. *P<0.001,
MPP group [2.45 (1.69-4.34)] vs. CAP with NMP group [1.01 (0.88-1.12)].
24
Author Contributions Conceived and designed the experiments: Lina Wu, Rui Zheng. Performed the experiments: Lina Wu, Maosheng Ye, Zhe Lv. Analyzed the data: Lina Wu, Xiaosong Qin. Contributed reagents/materials/analysis tools: Lina Wu, Yong Liu. Wrote the paper: Lina Wu, Rui Zheng.
25
26
27
28
29
30
Table 1
The clinical characteristics of the patients in the MPP, CAP with NMP, and control groups.
Parameters
CAP with NMP
MPP group
Age (years) a Gender (male:female)
c
Fever percentage before admission
group
P值 ＜0.001
39.98±12.86
62.58±15.74
39.47±13.79
83:79
117:111
80:82
0.930
------
＜0.001
------
＜0.001
------
＜0.001
------
0.032
82.72
48.25
% (n/N) c
(134/162)
(110/228)
Fever duration before admission (days) b
4.89±2.76
3.00±3.78
95.06
77.19
% (n/N) c
(154/162)
(176/228)
Cough duration before admission(days) b
8.29±2.91
7.44±4.83
Cough percentage before admission
Control group
MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group. Age (years), fever duration before admission (days) and cough duration before admission(days), all of them followed a normal distribution and were expressed as mean ± standard deviation. Fever percentage before admission and cough percentage before admission were presented as proportions, and in the brackets “n” represents the number of patients of fever or cough before admission, and “N” represents the total number of patients in each group. a comparisons made by analysis of variance; b comparisons made by t-test; c comparisons made by χ2 test. P < 0.05 was considered statistically significant.
Table 2
The positive rate of M. pneumoniae antibodies with PA in the MPP, CAP with NMP and control groups at admission.
Qualitative results Negative % (n/N)
Titer <1:40 1:40
Positive % (n/N)
1:80
≧1:160
MPP group
CAP with NMP group
Control group
12.35
55.26
55.56
(20/162)
(126/228)
(90/162)
22.84
25.00
22.22
(37/162)
(57/228)
(36/162)
25.93
13.60
14.81
(42/162)
(31/228)
(24/162)
38.88
6.14
7.41
(63/162)
(14/228)
(12/162)
P值 ＜0.001
0.86 0.02 ＜0.001
M. pneumoniae: Mycoplasma pneumoniae, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, PA: passive particle agglutination. Data are shown as percentages and analyzed using the χ2 test. In the brackets “n” represents the number of samples meeting the requirements of the titer in the second column, and “N” represents the total number of samples in each group. P < 0.05 was considered statistically significant.
31
Table 3
The ratios of 4 subgroups in the MPP, CAP with NMP groups during hospitalization
and control group. Blood collection
Subgroups % (n/N)
Groups
time
MP-IgM-/IgG-
MPP group At admission
7 days after admission
MP-IgM-/IgG+
MP-IgM+/IgG-
MP-IgM+/IgG+
19.14
62.35
1.23
17.28
(31/162)
(101/162)
(2/162)
(28/162)
CAP with NMP
35.09
64.47
0.00
0.44
group
(80/228)
(147/228)
(0/228)
(1/228)
0.00
67.90
0.00
32.10
(0/162)
(110/162)
(0/162)
(52/162)
MPP group CAP with NMP
39.47
60.53
0.00
0.00
group
(90/228)
(138/228)
(0/228)
(0/228)
Control group
19.14
80.86
0.00
0.00
(31/162)
(131/162)
(0/162)
(0/162)
P值 ＜0.001
＜0.001
＜0.001
＜0.001
＜0.001
MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, MP-IgM: Mycoplasma pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive. Data are shown as percentages and analyzed using the χ2 test. In the brackets “n” represents the number of samples meeting the requirements of each subgroup, and “N” represents the total number of samples in each group. P < 0.05 was considered statistically significant.
Table 4
In the light of the results of 3 subgroups in the MPP group by the PA test, the changes in the ELISA method among these patients.
Groups by PA results
Number of patients
Subgroup1
20
Results of ELISA method At admission MP-IgM-/IgG-
Result changing during hospitalization IgM/IgG seroconversion IgG seroconversion
MP-IgM-/IgG+.
10
IgG
MP-IgM+/IgG+
11
IgM/IgG seroconversion
MP-IgM-/IgG+
10
IgG seroconversion
MP-IgM+/IgG+
9
MP-IgM-/IgG+
82
MP-IgM+/IgG-
MP-IgM+/IgG+
2
MP-IgM+/IgG+
MP-IgM+/IgG+
24
MP-IgM+/IgG+
MP-IgM+/IgG+
4
MP-IgM-/IgG+ 138
4
Number of patients 2 8
MP-IgM-/IgG-
Subgroup3
days after admission MP-IgM+/IgG+ MP-IgM-/IgG+
MP-IgM-/IgG+
Subgroup2
7
IgM seroconversion & IgG (Data in Table S2) IgG IgG seroconversion & IgM+ (Data in Table S4) IgM+ & IgG+ (Data in Table S3) IgM+ & IgG+ (Data in Table S3)
MPP group: Mycoplasma pneumoniae pneumonia group, PA: passive particle agglutination, ELISA: enzyme-linked immunosorbent assays, MP-IgM: Mycoplasma pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive.
32
Highlights ● Quantitative ELISA MP-IgG was assessed for acute M. pneumoniae pneumonia diagnosis. ● Reference criterion was a single serum MP-IgG level of ≥ 92.67 RU/mL. ● Diagnostic criteria were ≥1.48-fold change for paired serum samples obtained within 7 days and values of
≥ 92.67 RU/mL after 7 d.
● Qualitative MP-IgM and quantitative MP-IgG detection by ELISA identifies adult MPP.
33
S0009-8981(20)30012-7 https://doi.org/10.1016/j.cca.2020.01.004 CCA 15984
To appear in:
Clinica Chimica Acta
Received Date: Revised Date: Accepted Date:
14 March 2019 7 January 2020 7 January 2020
Please cite this article as: L. Wu, M. Ye, X. Qin, Y. Liu, Z. Lv, R. Zheng, Diagnostic value of quantitative MPIgG for Mycoplasma pneumoniae pneumonia in adults, Clinica Chimica Acta (2020), doi: https://doi.org/ 10.1016/j.cca.2020.01.004
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© 2020 Published by Elsevier B.V.
Diagnostic value of quantitative MP-IgG for Mycoplasma pneumoniae pneumonia in adults Lina Wu1, Maosheng Ye2, Xiaosong Qin1, Yong Liu1, Zhe Lv1, Rui Zheng2* 1 Department of Laboratory Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China, 2 Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China *Corresponding author at: Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China
1
Abstract The passive particle agglutination (PA) test1, once widely used for Mycoplasma pneumoniae (M. pneumoniae) antibody detection, has gradually been replaced by quantitative enzyme-linked immunosorbent assays (ELISA). However, the lack of diagnostic criteria for quantitative ELISA M. pneumoniae-IgG(MP-IgG) and the low positive rates of ELISA M. pneumoniae-IgM (MP-IgM) limit the diagnostic value of ELISA for M. pneumoniae infection in adults. Here, the diagnostic value of quantitative ELISA MP-IgG was evaluated in adults with Mycoplasma pneumoniae pneumonia (MPP). The serum M. pneumoniae antibodies were detected in 162 patients with MPP, 228 patients with community-acquired pneumonia (CAP) with non-Mycoplasma pneumoniae(NMP), and 162 healthy controls by ELISA, using the PA results as the reference standards. For the MP-IgM-/IgG+ subgroup, a single serum MP-IgG level of ≥92.67 RU/mL can be used as a reference criterion for the diagnosis of acute M. pneumoniae infection. At admission, for patients with CAP, the sensitivity and specificity of ELISA MP-IgM positivity for MPP were 18.51% and 99.56%, respectively. MP-IgM positivity combined with MP-IgG ≥92.67 RU/mL increased the sensitivity to 40.12% and decreased the specificity to 94.29%. For paired serum samples obtained within seven days, an ELISA MP-IgG concentration change of ≥ 1.48-fold and MP-IgG ≥ 92.67 RU/mL on day 7 were used as the diagnostic criteria for M. pneumoniae infection. Accordingly, the combination of qualitative MP-IgM detection and quantitative MP-IgG detection by ELISA is
Abbreviations: PA , passive particle agglutination ; M. pneumoniae , Mycoplasma pneumoniae ; ELISA , enzyme-linked immunosorbent assays ; MPP , Mycoplasma pneumoniae pneumonia ; CAP , community-acquired pneumonia ; NMP, non-Mycoplasma pneumoniae; MP-IgG: M. pneumoniae-IgG; MP-IgM: M. pneumoniae-IgM Corresponding author at: Department of Pulmonary and Critical Care Medicine, Shengjing Hospital of China Medical University, Shenyang, PR China E-mail address: [email protected] 2
valuable for acute MPP diagnosis in adults.
3
Keywords: Mycoplasma pneumoniae; passive particle agglutination test; enzyme-linked immunosorbent assay; IgG; IgM
4
1. Introduction Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen leading to community-acquired pneumonia (CAP) in adults [1-4]. Owing to its lack of a cell wall, M. pneumoniae is intrinsically resistant to cell wall-targeting antibacterial drugs, such as β-lactams [5], and is only sensitive to fluoroquinolones, macrolides, and tetracyclines. To avoid treatment delay, an increasing number of patients with suspected M. pneumoniae infection are treated with macrolide antibiotics based on clinical symptoms and imaging findings during the early stage, despite the difficulty in differentiating Mycoplasma pneumoniae pneumonia (MPP) from CAP caused by other pathogens using this approach [6]. In addition, after the infections are cured, M. pneumoniae might remain in the respiratory tract mucosa [7-9]. As macrolides are widely used in clinical practice, the prevalence of macrolide resistance has also increased worldwide [10-12]. Compared with that of patients with macrolide-sensitive M. pneumoniae infection, patients with macrolide-resistant M. pneumoniae infection experience prolonged persistent cough, fever, and antibiotic treatment [13-18]. Therefore, it is essential to select the optimal laboratory tests and establish effective diagnostic criteria for obtaining a definitive diagnosis, initiating timely and effective treatment, and reducing the high rate of drug-resistance due to the misuse of antibiotics for patients infected with M. pneumoniae. A rapid and reliable serologic test for M. pneumoniae antibodies can provide strong evidence for diagnosis. The passive particle agglutination (PA) test is conventionally used to detect M. pneumoniae-specific antibodies in the serum, and its diagnostic value for M. pneumoniae infection has been demonstrated in clinical settings [1,19,20,21]. However, PA is prone to subjectivity with respect to the manual
5
procedures and interpretation of results. In addition, PA cannot differentiate between IgM and IgG antibodies. As is known to all, MP-IgM appears at the 1st week, and achieves the peak at 3 weeks after M. pneumoniae infection. MP-IgM positive is of important reference significance for the early or acute diagnosis of M. pneumoniae infection. Therefore, this test may be gradually replaced by quantitative enzyme-linked immunosorbent assays (ELISA). Compared with the PA test, the ELISA can be used to detect M. pneumoniae-IgM (MP-IgM) and M. pneumoniae-IgG (MP-IgG) antibodies, respectively. In addition, the use of an automated enzyme immunoassay analyzer enables easy MP-IgM and MP-IgG detection and the objective interpretation of results. This has improved the accuracy of test results and has almost overcome the limitations of PA. It has been reported that some adult patients with repeated M. pneumoniae infection have sustained MP-IgM negative [22]. In this study, the ratio of MP-IgM-/IgG+ subgroup was the highest in MPP, CAP with NMP, control groups; and the concentration of MP-IgG in the MPP group was significantly higher than that in the CAP with NMP and control groups at the same time during hospitalization. This indicates that the quantitative ELISA MP-IgG results will be of great value for the diagnosis of M. pneumoniae infection. However, as the results of ELISA are not expressed in titers but in S/CO or concentrations, the previous diagnostic criterion for the detection of M. pneumoniae infection by PA (a 4-fold or greater change in the titer between paired acute- and convalescent-phase serum samples) is not applicable [23,24]. Therefore, there is an urgent need to establish diagnostic criteria for the ELISA for M. pneumoniae infection. Lee et al. [25] have
6
suggested that for patients with severe conditions, especially for hospitalized patients, a titer change of 4-fold or greater within one week is helpful for the early diagnosis of M. pneumoniae infection. In the present study, an increase in paired serum titers of ≥4-fold within seven days or an initial serum titer of ≥1:640 with an increase of ≤2-fold by the PA test were used as the inclusion criterion for the MPP group of patients with CAP. We evaluated the diagnostic value and criteria of qualitative ELISA MP-IgM and quantitative ELISA MP-IgG for adults with MPP.
2. Materials and methods 2.1. Subjects Between November 2016 and June 2018, 390 patients with confirmed CAP in the Department of Pulmonary and Critical Care Medicine of Shengjing Hospital of China Medical University were included in the patient group. A total of 162 healthy individuals who visited the physical examination center and had no respiratory tract infection within the last six months with normal chest CT, white blood cell (WBC) count, and C-reactive protein (CRP) level were included in the control group. Mycoplasma pneumoniae infection was diagnosed based on the following internationally recognized criterion: a 4-fold or greater change in the serum M. pneumoniae-specific antibody titers from acute- and convalescent-phase paired serum samples [23, 24]. Patients with CAP (n = 390) were divided into the MPP group and the CAP with NMP group based on the changes in serum M. pneumoniae antibody titers detected by PA between the time of admission and seven days after admission. The MPP group was further divided into three subgroups according to the changes in M. pneumoniae antibodies detected by PA. Subgroup 1 included 20 patients with 7
seroconversion (i.e., negative for M. pneumoniae antibodies at admission, with a titer of ≥ 1:160 seven days after admission). Subgroup 2 included 138 patients whose M. pneumoniae antibody titers increased to ≥ 4-fold within seven days. Subgroup 3 included four patients with M. pneumoniae antibody titers of ≥1:640 at admission, with a change in M. pneumoniae antibody titers of ≤ 2-fold within seven days . Patients with CAP who did not meet the above criteria were assigned to the CAP with NMP group. Clinical characteristics, including age, gender, and symptoms of patients in the MPP, CAP with NMP, and healthy control groups are shown in Table 1; Age distribution of each group is detailed in Table S1. Fasting venous blood (2 mL) was drawn from patients in the MPP and CAP with NMP groups at admission and seven days after admission. The blood samples were centrifuged at 1200g for10 minutes, and the serum was separated and stored in a -70°C freezer. The leftover serum from healthy individuals after physical examination was collected and stored in a -70°C freezer. All samples were collected and tested centrally in one laboratory. This study was approved by the ethics committee (approval number: 2018PS429K) of Shengjing Hospital of China Medical University.
2.2. Methods PA was used to detect M. pneumoniae antibodies. The ELISA was used to detect MP-IgM and MP-IgG antibodies. Reagents for PA (SERODIA®-MYCOII) and the ELISA were purchased from FUJIREBIO Co., Ltd. (Tokyo, Japan) and EUROIMMUN Medical Laboratory Diagnostics (Lubeck, Germany), respectively. PA was used to detect a mixture of M. pneumoniae antibodies and the results are expressed as follows: negative (titer: <1:40) and positive (titers: 1:40, 1:80, 1:160, 1:320, 1:640, and ≥1:1280). In order to ensure the accuracy of M. pneumoniae antibody with PA, the whole operation was carried out by training and assessing 8
technicians according to the standard operating procedure of M. pneumoniae antibody with PA. Each test result will be interpreted separately by two technicians. If consistent, the result will be reported to the clinic. But if not, the result will be made a check by the competent technician or re-test if necessary. The ELISA was used to detect MP-IgM and MP-IgG antibodies by TECAN automated enzyme immunoassay analyzer . According to the manufacturer’s protocol of the ELISA (EUROIMMUN Medical Laboratory Diagnostics, Lubeck, Germany) , the results of ELISA MP-IgM based on S/CO were interpreted as follows: negative if <0.8, equivocal if 0.8–1.1, and positive if ≥1.1. The results of ELISA MP-IgG were interpreted as follows: negative if <16 RU/mL, equivocal if 16–22 RU/mL, and positive if ≥22 RU/mL. For both ELISA MP-IgM and MP-IgG, samples with results within the equivocal range were tested at least twice again. If the coefficient of variation of multiple test results is less than 10% and not more than 1.1 times of the precision within-run of the corresponding concentration provided by the kit instruction, the mean of the multiple test results is taken to judge the negative or positive results. If the mean is still in the equivocal range or positive, it is counted as positive; otherwise, negative.
2.3. Statistical analysis Qualitative data are expressed as percents and were analyzed using the χ2 test. Quantitative data that followed a normal distribution were expressed as mean ± —
standard deviation (X ± S) and were analyzed using the t-test or analysis of variance. Quantitative data that did not follow a normal distribution were expressed as median (interquartile range) and were analyzed using the Kruskal–Wallis H test. Correlation analyses were conducted using linear regression. All statistical analyses were performed using SPSS 20.0. Differences with P < 0.05 were considered statistically 9
significant.
3. Results 3.1. At admission, both the positive rate and titer in MPP group as determined by the PA test were significantly higher than those in the CAP with NMP group and the control group Based on a qualitative analysis using 1:40 as the cut-off value for M. pneumoniae infection, the positive detection rate for the MPP, CAP with NMP, and control groups was 87.65%, 44.74%, and 44.44%, respectively, and the positive rate in the MPP group was significantly higher than that in the CAP with NMP and control groups (Table 2, P ＜0.001). For a titer of ≥1:160, the positive rate in the MPP group (38.88%) was significantly higher than that in the CAP with NMP(6.14%) and control groups (7.41%) (Table 2, P＜0.001). At admission, the median titer in the MPP group (1:80) was significantly higher than that in the CAP with NMP (<1:40) and control groups (<1:40) (Figure 1, P＜0.001), and there was no statistical difference between the CAP with NMP and control group (Figure 1). 3.2. The proportion of ELISA MP-IgG single positive patients was the highest in the MPP, CAP with NMP groups during hospitalization and control group According to the qualitative results for MP-IgM and MP-IgG obtained by the ELISA at admission, patients in the three groups could be divided into the following four subgroups: IgM and IgG positive (MP-IgM+/IgG+), IgM single positive (MP-IgM+/IgG-), IgG single positive (MP-IgM-/IgG+), and IgM and IgG negative (MP-IgM-/IgG-). In the MPP group, the CAP with NMP group during hospitalization, and the control group, the MP-IgM-/IgG+ subgroup was the most frequent among the four subgroups (Table 3, P＜0.001). 3.3. For the MP-IgM-/IgG+ subgroup, the level of MP-IgG in the MPP group was 10
significantly higher than that in CAP with NMP and control groups At admission, the quantitative results for the MP-IgG in MP-IgM-/IgG+ subgroup showed that the median level of MP-IgG in the MPP group [ n = 101, 59.74 (33.01-96.96) RU/mL] was significantly higher than that in the CAP with NMP [ n = 147, 39.69 (23.34-61.43) RU/mL, P < 0.001] and control groups [n = 131, 50.00 (28.35-71.56) RU/mL, P = 0.041] (Figure 2A). At seven days after admission, the median level of MP-IgG in the MPP group [n = 110, 156.84 (138.48-187.77) RU/mL] was significantly higher than that in the CAP with NMP [n = 138, 45.07 (26.48-66.66) RU/mL, P < 0.001] and control groups [n = 131, 50.00 (28.35-71.56) RU/mL, P < 0.001] (Figure 2B). 3.4. For MP-IgG single positive patients, the concentration of ELISA MP-IgG was 92.67RU/mL, which may be used as a reference criterion to the diagnosis of M. pneumoniae infection Similar to the titer change in paired serum samples obtained by the PA test for the MPP group, the ELISA also showed seroconversion and increase in antibody concentrations (Table 4). To explore the correlation between the concentration of MP-IgG obtained by the ELISA and the titers obtained by the PA test, we evaluated patients who were MP-IgM-negative during hospitalization in the MPP group (n = 110, specimens = 220, Table 4), CAP with NMP group (n = 227, specimens = 454, Table 3), and MP-IgM-negative controls (n = 162, specimens = 162, Table 3). The ELISA MP-IgG levels were highly correlated with the PA titers (Y = 24.85 + 23.74X, F = 1149.91, where Y and X represent the concentration of MP-IgG by the ELISA and PA titer, respectively, and the titers obtained by the PA test (from <1:40 to 1:10240) were replaced with a value of 0 to 9, P < 0.001) (Figure 3). Corresponding to a titer of 1:160 using the PA test, the 95% confidence interval of MP-IgG levels determined by 11
the ELISA was 92.67 - 99.47 RU/mL. 3.5. For MP-IgM-/IgG+ subgroup, an ELISA MP-IgG concentration change of ≥ 1.48-fold can be used as an exact criterion to the diagnosis of M. pneumoniae infection To explore the MP-IgG concentration change in the acute phase of M. pneumoniae infection by the ELISA, we evaluated patients with seroconversion or increased antibody titers (≧4) using the PA test and solitary ELISA MP-IgG-positive during hospitalization in the MPP group (n = 92, Table 4). The median and one-sided 95% confidence interval of MP-IgG increase in paired serum samples by the ELISA were 2.45 (1.69-4.34) (Figure 4) and ≧1.48, respectively. Furthermore, for patients identified as solitary IgG-positive during hospitalization in the CAP with NMP group (n = 137), the median and one-sided 95% confidence interval for increase in ELISA MP-IgG were 1.01 (0.88-1.12) (Figure 4) and ≤1.53, respectively .
4. Discussion In this study, PA was used as the reference standard to investigate the diagnostic value and threshold criteria for ELISA MP-IgM and MP-IgG in adults with MPP. For MPP diagnosis in adults with CAP, at admission and on day 7 after admission, the diagnostic sensitivity of ELISA MP-IgM positivity were 18.51% and 32.10% (Table S5), respectively. This indicates that ELISA MP-IgM positivity alone is insufficient for the diagnosis of M. pneumoniae infection in adults. The subgroup with single positive results for ELISA MP-IgG accounted for the highest proportion of patients in the MPP, CAP with NMP, and control groups; at admission and on day 7 after admission, the concentration of MP-IgG in the MPP group was significantly higher than that in the CAP with NMP and control groups at the same time points. This suggests that the quantitative MP-IgG results obtained by the ELISA are of great 12
value for the diagnosis of M. pneumoniae infection. Further investigations showed that in the MP-IgG-single positive subgroup, a single serum ELISA MP-IgG level of ≥ 92.67 RU/mL can be used as a reference criterion for the diagnosis of M. pneumoniae infection, whereas a change in the concentration of MP-IgG by ELISA within seven days of ≥1.48-fold in the paired serum samples and MP-IgG ≥92.67 RU/mL on day 7 after admission can be used as the diagnostic criteria for acute M. pneumoniae infection. Although a positive MP-IgM result for a single serum sample can be used as a diagnostic reference for M. pneumoniae infection [23], it does not provide a sufficient basis for the diagnosis of M. pneumoniae infection in adults. The positive detection rate of MP-IgM in the MPP group in the present study was consistent with the results of Martinez et al., who showed that the diagnostic sensitivity of MP-IgM for acute M. pneumoniae infection in adults was 33.3% [26]. It has been reported that the positive detection rate of MP-IgM is related to age. Chamberlain et al. performed a complement fixation test and found that the positive rate of MP-IgM in patients with MPP under 20 years of age was approximately 78%, whereas that in adult patients was only 34% [27]. Moule et al. [28] performed ELISA (μ-capture) and found that approximately 91% of patients under the age of 20 were positive for MP-IgM, whereas the positive rate in adult patients was approximately 50%. Initial M. pneumoniae infection can lead to the production of memory B cells and T cells, which are rapidly activated upon reinfection. This is manifested in some patients as a rise in MP-IgM after 2-3 days of infection, followed by a peak in the MP-IgM level within a short period and a rapid increase in IgG thereafter; other patients may show an immediate rise in the IgG level. Therefore, we speculate that the differences in the positive detection rate of MP-IgM among age groups following M. pneumoniae
13
infection may be related to differences in the proportion of patients re-infected with M. pneumoniae. Luciani L et al have defined residual antibodies as low or intermediate rate of IgG against Coxialla burnetii that can persist for several months or even years after primary infection, without active infection.Therefore, IgG is a well-established indicator of previous infection [29]. In this study, the high positive rate of MP-IgG in the MPP, CAP with NMP groups during hospitalization and control group suggest a high proportion of adults with M. pneumoniae reinfection. Specifically, in the MPP group, 101 and 110 patients were MP-IgG single positive at admission and seven days after admission, accounting for 62.35% and 67.90%, respectively; 7 days after admission, 9 patients with MP-IgG single positive at admission converted to MP-IgM+/MP-IgG+, and 18 patients with MP-IgM-/MP-IgGat admission converted to MP-IgG single positive. In the CAP with NMP group, 147 and 138 patients were MP-IgG single positive at admission and seven days after admission, accounting for 64.47% and 60.53%, respectively; 7 days after admission, 10 patients with MP-IgG single positive at admission converted to MP-IgM-/MP-IgG-, and 1 patients with MP-IgM+/MP-IgG+ at admission converted to MP-IgG single positive. In view of the high proportion of adults with M. pneumoniae reinfections and assuming that the time from symptom onset to hospital visit is generally longer than one week, the scientific use of quantitative MP-IgG results as a reference for the diagnosis of M. pneumoniae infection may compensate for the diagnostic limitations of MP-IgM, to some extent. Yoshimasu et al. [30] have reported that a high titer of anti-Helicobacter pylori IgG antibodies may be an indicator for upper gastrointestinal endoscopy and eradication therapy in patients with acute or chronic urticaria. Sonmez et al. [31] have proposed that the anti-PT IgG antibody levels of ≥100 EU/mL should
14
be considered as an indicator of acute or recent infection with Bordetella pertussis. IgG and IgM are the most important antibodies used in serologic testing for M. pneumoniae infection. Owing to the potential lack of an IgM response in adults re-infected with M. pneumoniae, the IgG level is particularly important for the diagnosis of M. pneumoniae reinfection [32]. In the present study, in the subgroup of patients positive for MP-IgG only, both at admission and seven days after admission, the median MP-IgG concentration in the MPP group was significantly higher than that in the CAP with NMP and control groups. And in the MPP group, the MP-IgG concentration on seven days after admission was significantly higher than that at admission (P ＜ 0.001, Data in Figure 2A and Figure 2B). However, there was no significant difference in MP-IgG concentration between the two time points in the CAP with NMP group (P = 0.318, Data in Figure 2A and Figure 2B) . This suggests that the concentration of MP-IgG can be used as a reference for the diagnosis of acute M. pneumoniae infection. There is a consensus in available literature that a single serum M. pneumoniae antibody titer of ≥ 1:160 detected by PA should be used as a reference for the diagnosis of recent M. pneumoniae infection [22]. For patients who were MP-IgM-negative during hospitalization in the MPP, CAP with NMP, and the control groups, we found that the level of MP-IgG determined by the ELISA was correlated with the PA titers. Corresponding to a titer of 1:160 for the PA test, the 95% confidence interval of MP-IgG by the ELISA was 92.67 - 99.47 RU/mL. The combination of MP-IgM positivity and MP-IgG ≥ 92.67 RU/mL as a reference for the diagnosis of acute M. pneumoniae infection can significantly improve the diagnostic sensitivity and positive predictive value for M. pneumoniae infection. When the two thresholds were combined, the sensitivity and specificity for the diagnosis of MPP in patients with CAP at admission were 40.12% and 94.29%
15
(Table S6), respectively, which were slightly higher than those for a titer of 1:160 by PA (i.e., 38.88% and 93.86%, Table S7). When compared with the positivity for MP-IgM alone (18.51% and 99.56%), the diagnostic sensitivity improved significantly, while the diagnostic specificity decreased. According to the health industry standard of the People’s Republic of China, WS/T 494-2017 “Guideline for performance characteristics of immunological qualitative test”, the diagnostic tests are used to detect the presence of clinically suspected analytes, such as infectious pathogen antigens or related antibodies. If the infective pathogen antigen or related antibody to be tested is important for treatment and prognosis, the diagnostic test should be sensitive enough on the premise of ensuring certain clinical specificity (≥ 90%) [33]. As a diagnostic test for acute or recent infection of M. pneumoniae, the results of serum M. pneumoniae antibody are very important for scientific selection of treatment options and reduction of M. pneumoniae with macrolides resistant. The serological test of M. pneumoniae antibody should be sensitive enough on the premise of ensuring certain clinical specificity ( ≥ 90%). Therefore, The combination of MP-IgM positivity and MP-IgG ≥ 92.67 RU/mL is more suitable as a reference standard for acute or recent M. pneumoniae infection. Clinicians prefer the use of positive predictive value (PPV) and negative predictive value (NPV) over diagnostic sensitivity and specificity. Based on the sensitivity and specificity of the ELISA and PA as well as the assumption that the incidence of MPP in patients with CAP was 30%, the combination of MP-IgM positivity and MP-IgG ≥92.67 RU/mL would correspond to a PPV and NPV of 75.07% and 78.61% (Table S8), respectively, both of which were higher than those for PA (PPV: 73.07%, NPV: 78.18%, Table S9). In addition to the high proportion of patients positive for MP-IgG only at admission, this study also included a certain proportion of patients who were negative
16
for both IgM and IgG. To avoid missed diagnosis, we collected paired serum samples from patients during hospitalization and found that 31 patients in the MPP group were negative for both IgM and IgG at admission. At seven days after admission, 13 patients presented positive for both IgM and IgG (six patients with MP-IgG < 92.67 RU/mL), and 18 patients had significant seroconversion for IgG only (ten patients with MP-IgG < 92.67 RU/mL). The occurrence of seroconversion supports the diagnosis of acute M. pneumoniae infection. However, for the subgroup of patients positive for MP-IgG only during hospitalization, the proper use of a concentration change in the paired serum samples for the diagnosis of M. pneumoniae infection is a major concern for clinicians.As mentioned above, a change in titers of ≥ 4-fold between the acute and convalescent phases is a definitive criterion for the diagnosis of M. pneumoniae infection [23,24]; however, this criterion is based on qualitative and visual assessments of the PA and ELISA results. This criterion enables a preliminarily determination of the change in antibody concentration by gradually diluting the sample, under the premise that the antibody concentration itself cannot be accurately detected. Owing to the development of automated enzyme immunoassay analyzers in the 1990s, the current ELISA kits no longer use titers to determine results, but rather directly calculate the S/CO values or antibody concentrations in the serum using microplate readers to obtain semiquantitative and quantitative results. Therefore, the above diagnostic criterion no longer applies to the current ELISA kits. Loens et al. [34] suggested that the clinical significance of serologic tests for IgG should be determined by studies on patients with documented infection and detailed information on the time lapse between disease onset and the collection of serum specimens. Therefore, to investigate the changes in serum MP-IgG concentrations detected by the ELISA in the acute phase of M. pneumoniae infection, we enrolled 92
17
patients in the MPP group whose paired serum samples collected during hospitalization showed seroconversion or an antibody titer increase of ≥4-fold as detected by the PA test, with positive results for ELISA MP-IgG only. In a comparative analysis, the one-sided 95% confidence interval for concentration changes detected in paired serum samples by ELISA MP-IgG in the MPP and CAP with NMP groups was ≥1.48- and ≤1.53-fold, respectively. This suggests that a concentration change in paired serum MP-IgG between 1.48 and 1.53 is a gray area for diagnosis. Further analysis of the 17 patients in CAP with NMP group whose change in MP-IgG concentration was ≥1.48 revealed that the MP-IgG concentration at admission was between 16.00 and 30.00 RU/mL and the MP-IgG concentration increased seven days after admission to between 24.00 and 60.00RU/mL. These concentrations were lower than 92.67 RU/mL at both time points. Conversely, the MP-IgG concentration in all 92 patients in the MPP group was > 92.67 RU/mL at seven days after admission. Further investigations of patients in the MPP group whose change in the paired sera concentration within seven days was <1.48 showed that the MP-IgG concentration was approximately 200 RU/mL in all patients during hospitalization. The upper limit of quantification for the ELISA MP-IgG kit used in this study was 200 RU/mL; the validity of these results as diagnostic criteria for M. pneumoniae infection could not been confirmed owing to the limited number of cases in this study. Therefore, the following can be used as diagnostic criteria for acute M. pneumoniae infection in adults with positive ELISA MP-IgG only: ≥1.48-fold difference in the MP-IgG concentration within seven days and an MP-IgG concentration in the second serum sample of ≥ 92.67 RU/mL. In summary, the combination of qualitative MP-IgM detection and quantitative MP-IgG detection by ELISA is of significant importance for the diagnosis of MPP in
18
adults. To date, quantitative ELISA MP-IgG has not been used as a reference standard and diagnostic criterion for the diagnosis of MPP in adults. Nevertheless, M. pneumoniae antigens from different manufacturers have different origins and traceability standards. Therefore, a limitation of the study is that the results are only applicable to the ELISA kits with the same traceability as that of the kit used in this study. Acknowledgments We would like to express our gratitude to all of the subjects who participated in this study. Potential conflict of interest We declare that no competing interests exist. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions Conceived and designed the experiments: Lina Wu, Rui Zheng. Performed the experiments: Lina Wu, Maosheng Ye, Zhe Lv. Analyzed the data: Lina Wu, Xiaosong Qin. Contributed reagents/materials/analysis tools: Lina Wu, Yong Liu. Wrote the paper: Lina Wu, Rui Zheng.
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Figure Legends Figure 1. The titers of M. pneumoniae antibodies with PA in the MPP, CAP with NMP and control groups at admission. M. pneumoniae: Mycoplasma pneumoniae, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, PA: passive particle agglutination. Data was expressed as median, and analyzed using the Kruskal–Wallis H test. P < 0.05 was considered statistically significant. *P<0.001,
MPP group vs. CAP with NMP group；#P<0.001, MPP group vs. control group
Figure 2. The concentration of ELISA MP-IgG in the MP-IgM-/IgG+ subgroup in the MPP, CAP with NMP groups during hospitalization and control group. MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, ELISA: enzyme-linked immunosorbent assays, MP-IgM: Mycoplasma 23
pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive. Data was expressed as median (interquartile range), and analyzed using the Kruskal–Wallis H test. P < 0.05 was considered statistically significant. A. at admission:*P<0.001, MPP group [59.74 (33.01-96.96) RU/mL] vs. CAP with NMP group [39.69 (23.34-61.43) RU/mL];
#P=0.041,
MPP group [59.74 (33.01-96.96) RU/mL] vs. control group [50.00
(28.35-71.56) RU/mL]. B. 7 days after admission: *P<0.001, MPP group [156.84 (138.48-187.77) RU/mL] vs. CAP with NMP group [45.07 (26.48-66.66) RU/mL]; #P<0.001, MPP group [156.84 (138.48-187.77) RU/mL] vs. control group [50.00 (28.35-71.56) RU/mL].
Figure 3. The correlation between the concentration of MP-IgG in ELISA and the titer of PA. M. pneumoniae: Mycoplasma pneumoniae, PA: passive particle agglutination, ELISA: enzyme-linked immunosorbent assays, MP-IgG: Mycoplasma pneumoniae-IgG. The titers of PA from ＜ 1:40 to 1:10240 were replaced by 0 to 9 respectively, and the equation of regression was Y=24.85+23.74X, F=1149.91, P ＜0.001.
Figure 4. The increasing times of MP-IgG with ELISA within 7 days in MPP and CAP with NMP groups. ELISA: enzyme-linked immunosorbent assays, MP-IgG: Mycoplasma pneumoniae-IgG, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group. *P<0.001,
MPP group [2.45 (1.69-4.34)] vs. CAP with NMP group [1.01 (0.88-1.12)].
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Author Contributions Conceived and designed the experiments: Lina Wu, Rui Zheng. Performed the experiments: Lina Wu, Maosheng Ye, Zhe Lv. Analyzed the data: Lina Wu, Xiaosong Qin. Contributed reagents/materials/analysis tools: Lina Wu, Yong Liu. Wrote the paper: Lina Wu, Rui Zheng.
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Table 1
The clinical characteristics of the patients in the MPP, CAP with NMP, and control groups.
Parameters
CAP with NMP
MPP group
Age (years) a Gender (male:female)
c
Fever percentage before admission
group
P值 ＜0.001
39.98±12.86
62.58±15.74
39.47±13.79
83:79
117:111
80:82
0.930
------
＜0.001
------
＜0.001
------
＜0.001
------
0.032
82.72
48.25
% (n/N) c
(134/162)
(110/228)
Fever duration before admission (days) b
4.89±2.76
3.00±3.78
95.06
77.19
% (n/N) c
(154/162)
(176/228)
Cough duration before admission(days) b
8.29±2.91
7.44±4.83
Cough percentage before admission
Control group
MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group. Age (years), fever duration before admission (days) and cough duration before admission(days), all of them followed a normal distribution and were expressed as mean ± standard deviation. Fever percentage before admission and cough percentage before admission were presented as proportions, and in the brackets “n” represents the number of patients of fever or cough before admission, and “N” represents the total number of patients in each group. a comparisons made by analysis of variance; b comparisons made by t-test; c comparisons made by χ2 test. P < 0.05 was considered statistically significant.
Table 2
The positive rate of M. pneumoniae antibodies with PA in the MPP, CAP with NMP and control groups at admission.
Qualitative results Negative % (n/N)
Titer <1:40 1:40
Positive % (n/N)
1:80
≧1:160
MPP group
CAP with NMP group
Control group
12.35
55.26
55.56
(20/162)
(126/228)
(90/162)
22.84
25.00
22.22
(37/162)
(57/228)
(36/162)
25.93
13.60
14.81
(42/162)
(31/228)
(24/162)
38.88
6.14
7.41
(63/162)
(14/228)
(12/162)
P值 ＜0.001
0.86 0.02 ＜0.001
M. pneumoniae: Mycoplasma pneumoniae, MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, PA: passive particle agglutination. Data are shown as percentages and analyzed using the χ2 test. In the brackets “n” represents the number of samples meeting the requirements of the titer in the second column, and “N” represents the total number of samples in each group. P < 0.05 was considered statistically significant.
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Table 3
The ratios of 4 subgroups in the MPP, CAP with NMP groups during hospitalization
and control group. Blood collection
Subgroups % (n/N)
Groups
time
MP-IgM-/IgG-
MPP group At admission
7 days after admission
MP-IgM-/IgG+
MP-IgM+/IgG-
MP-IgM+/IgG+
19.14
62.35
1.23
17.28
(31/162)
(101/162)
(2/162)
(28/162)
CAP with NMP
35.09
64.47
0.00
0.44
group
(80/228)
(147/228)
(0/228)
(1/228)
0.00
67.90
0.00
32.10
(0/162)
(110/162)
(0/162)
(52/162)
MPP group CAP with NMP
39.47
60.53
0.00
0.00
group
(90/228)
(138/228)
(0/228)
(0/228)
Control group
19.14
80.86
0.00
0.00
(31/162)
(131/162)
(0/162)
(0/162)
P值 ＜0.001
＜0.001
＜0.001
＜0.001
＜0.001
MPP group: Mycoplasma pneumoniae pneumonia group, CAP with NMP group: community-acquired pneumonia with non-Mycoplasma pneumoniae group, MP-IgM: Mycoplasma pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive. Data are shown as percentages and analyzed using the χ2 test. In the brackets “n” represents the number of samples meeting the requirements of each subgroup, and “N” represents the total number of samples in each group. P < 0.05 was considered statistically significant.
Table 4
In the light of the results of 3 subgroups in the MPP group by the PA test, the changes in the ELISA method among these patients.
Groups by PA results
Number of patients
Subgroup1
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Results of ELISA method At admission MP-IgM-/IgG-
Result changing during hospitalization IgM/IgG seroconversion IgG seroconversion
MP-IgM-/IgG+.
10
IgG
MP-IgM+/IgG+
11
IgM/IgG seroconversion
MP-IgM-/IgG+
10
IgG seroconversion
MP-IgM+/IgG+
9
MP-IgM-/IgG+
82
MP-IgM+/IgG-
MP-IgM+/IgG+
2
MP-IgM+/IgG+
MP-IgM+/IgG+
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MP-IgM+/IgG+
MP-IgM+/IgG+
4
MP-IgM-/IgG+ 138
4
Number of patients 2 8
MP-IgM-/IgG-
Subgroup3
days after admission MP-IgM+/IgG+ MP-IgM-/IgG+
MP-IgM-/IgG+
Subgroup2
7
IgM seroconversion & IgG (Data in Table S2) IgG IgG seroconversion & IgM+ (Data in Table S4) IgM+ & IgG+ (Data in Table S3) IgM+ & IgG+ (Data in Table S3)
MPP group: Mycoplasma pneumoniae pneumonia group, PA: passive particle agglutination, ELISA: enzyme-linked immunosorbent assays, MP-IgM: Mycoplasma pneumoniae-IgM, MP-IgG: Mycoplasma pneumoniae-IgG, -: negative, +: positive.
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Highlights ● Quantitative ELISA MP-IgG was assessed for acute M. pneumoniae pneumonia diagnosis. ● Reference criterion was a single serum MP-IgG level of ≥ 92.67 RU/mL. ● Diagnostic criteria were ≥1.48-fold change for paired serum samples obtained within 7 days and values of
≥ 92.67 RU/mL after 7 d.
● Qualitative MP-IgM and quantitative MP-IgG detection by ELISA identifies adult MPP.
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