Comparison of different detection methods for Mycoplasma pneumoniae infection in children with community-acquired pneumonia

doi:10.21203/rs.3.rs-29798/v2

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Comparison of different detection methods for Mycoplasma pneumoniae infection in children with community-acquired pneumonia

https://doi.org/10.21203/rs.3.rs-29798/v2

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published 19 Feb, 2021

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Background: Due to the lack of a sensitive, specific and rapid detection method, etiological diagnosis of pneumonia caused by Mycoplasma pneumoniae (M. pneumoniae, MP) is a constantly challenging issue. This retrospective study aimed to compare the diagnostic methods for mycoplasma pneumoniae in children and evaluate their values.

Methods: From November 2018 to June 2019, 830 children with community-acquired pneumonia were selected from the Department of Respiratory Medicine, Shanghai Children's Medical Center. On the first day of hospitalization, sputum, throat swabs, venous blood samples were collected for MP-IgM (particle agglutination, PA), MP-IgM (immune colloidal gold technique, GICT), MP-DNA, MP-RNA (simultaneous amplification and testing, SAT), and MP-DNA (real-time polymerase chain reaction, RT-PCR)

Results: Among these 830 children, RT-PCR showed that the positive rate was 36.6% (304/830), in which the positive rate of macrolide-resistant (A2063G mutation) accounted for 86.2% (262/304). Using RT-PCR as the standard, MP-RNA (SAT) had the highest specificity (97.5%), and MP-IgM (PA) had the highest sensitivity (74.0%) and Youden index (53.7%). If MP-RNA (SAT) was combined with MP-IgM (PA), its Kappa value (0.602), sensitivity (84.2%), specificity (78.7%), Youden index (62.9%), were higher than those of single Mycoplasma pneumoniae detection.

Conclusions: Our research indicated that a combination of MP-RNA (SAT) plus MP-IgM (PA) as an early diagnostic method for children with clinical manifestations of Mycoplasma pneumoniae pneumonia might lead to reliable results.

Pediatrics

Detection methods

Mycoplasma pneumoniae

Children

Community-acquired pneumonia

Mycoplasma pneumoniae (M. pneumoniae, MP) is known as a common cause of community-acquired pneumonia (CAP) in children throughout the world. It is the smallest prokaryotic microorganism, which lacks a cell wall and has a high affinity for respiratory epithelial cells. 40% or more cases of CAP in children are caused by M. pneumoniae and about 18% cases requiring hospitalization.¹ At present, it is believed that the pathogenic mechanism of M. pneumoniae is to cause direct injury through the community-acquired respiratory distress syndrome (CARDS) toxin, which induced eosinophilia, increased cytokine production, and hyperreactivity of the airway like asthma in animal models.^2-4 In addition, the immunological response resulting from infection by M. pneumoniae causes pulmonary and extrapulmonary symptoms.⁵

A worldwide increase in the prevalence of macrolide-resistant mycoplasma pneumoniae (MRMP) strains has been witnessed since 2000.⁶ In some regions of Asia, the resistance rates were reported over 90%,⁷ whereas in North America and Europe the rates reached 25%.^8,9 Compared with other patients with CAP, children with M. pneumoniae infection may be older and prolong the symptoms of fever and cough.¹⁰ Period of signs and symptoms will be shortened if accuracy antimicrobial treatment is started early in the course of diseases. However, Mycoplasma pneumoniae Pneumonia (MPP) in children cannot be diagnosed exactly in terms of clinical manifestations.¹¹

Mycoplasma pneumoniae Pneumonia has the characteristics of insidious onset, mild pulmonary signs, low specificity of clinical manifestations and image. Therefore, effective and sensitive laboratory diagnostic methods are the main basis for the diagnosis of M. pneumoniae infection. At present, there are many clinical methods for detecting M. pneumoniae, such as culture, serology and molecular-based methods. Though as the gold standard, culture is limited in clinical utility because of its time-consuming and insensitive.¹² And both serology and molecular assays have their own advantages and disadvantages in clinical practice. In this paper, we aimed to evaluate the clinical application value on different detection methods of M. pneumoniae infection by comparing and analyzing, in order to improve diagnostic efficiency.

Study population

We conducted a retrospective study including children aged 1 month to 15 years with radiologically confirmed community-acquired pneumonia. Venous blood, throat swabs and sputum specimens were obtained from these patients on the day of hospitalization at the Department of Respiratory Medicine, Shanghai Children’s Medical Center (SCMC) from November 1, 2018 to June 30, 2019. These specimens were tested for the MP-IgM (particle agglutination, PA), MP-IgM (immune colloidal gold technique, GICT), MP-RNA (simultaneous amplification and testing, SAT), 7 items of respiratory tract RNA (7 RNAs), and MP-DNA (real-time polymerase chain reaction, RT-PCR).

MP-IgM (particle agglutination, PA)

Particle agglutination (PA) antibody titers for M. pneumonia were assayed by using SERODIA MYCO-II (Fuji Rebio Ltd, Tokyo, Japanese), which were performed using artificial gelatin particles, sensitized with cell membrane components of M. pneumoniae. The result would be positive if the titer was 1:160 or more (≥1:160).

MP-IgM (immune colloidal gold technique, GICT)

According to the manufacturer’s (Alere Diagnostics, Shanghai, China) instructions, colloidal gold was used as a tracer marker to detect M. pneumoniae antibodies using the principle of immunoreaction of specific antigen-antibody. The results of this method showed only negative, weak positive or positive, without titers.

MP-RNA (simultaneous amplification and testing, SAT)

The throat swab specimens of the children were collected and tested by using the M. pneumoniae nucleic acid detection kits (Rendu Biotechnology, Shanghai, China) including nucleic acid extraction and isothermal amplification detection.

7 items of respiratory tract RNA (7 RNAs)

Based on the double-amplification method of RNA isothermal amplification and multi-biotin signal, throat swabs could be collected to find out seven common respiratory pathogen RNAs (Zhongzhi Biotechnologies, Wuhan, China), such as influenza A, influenza B, respiratory syncytial virus (RSV), parainfluenza virus, adenovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae, in a short period of time.

MP-DNA (real-time polymerase chain reaction, RT-PCR)

According to the specific point mutations in domain V of MP 23s rRNA (at positions 2063, 2064), the primers were designed as follows: Forward primer: 5´-AACTATAACGGTCCTAAGGTAGCG-3´; Reverse primer: 5´-GCTCCTACCTATTCTCTACATGAT-3´. The sputum sucked from the patients was collected for sample processing, DNA extraction with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), PCR reaction, and gel electrophoresis on the PCR product. Finally, the positive specimen was sequenced and then analyzed by using Snapgene software. The measured sequence was compared to the standard sequence of the M129 strain in the NCBI database. If the sequence was not consistent, A2063G transition could be found that was involved in macrolide-resistant. Unfortunately, we did not find a mutation at 2064.

Statistical analysis

SPSS software package v21.0 was used for all statistical analyses. Categorical variables were expressed as frequencies and percentages. Four-table diagnostic tests were used to calculate Kappa value, sensitivity, specificity, and Youden index. In a previous report,¹³ the value of the Kappa statistic was interpreted as follows: <0.01, poor; 0 to 0.20, slight; 0.21 to 0.4, fair; 0.41 to 0.6, moderate; 0.61 to 0.80, substantial; and 0.81 to 1, almost perfect. Sensitivity = true positive cases / (true positive cases + false negative cases) × 100%; Specificity = true negative cases / (true negative cases + false positive cases) × 100%; Youden index = sensitivity + specificity −1. Youden index could be used to evaluate the effectiveness and authenticity of experiments.

Clinical characteristics of patients

A total of 830 patients diagnosed with community-acquired pneumonia, aged 1 month to 15 years, were enrolled in the present study between November 1, 2018, and June 30, 2019. Congenital heart disease, congenital biliary atresia, epilepsy, hydrocephalus and cerebral palsy were the most frequently observed underlying diseases in these patients (Table 1).

Comparison of positive rates of M. pneumoniae detection methods in different age groups

All the patients were grouped by age as follows: infants (age: < 3 years), preschoolers (age: 3-5 years) and school-aged children (age: 6-15 years). The highest specimen positivity rate was in the school-aged children. And there were significant differences in the positivity rate of the different age groups by each detection methods except 7 RNAs (Table 2). In addition, the rate of macrolide-resistance was highest in school-age children (87.9%, 123/140) and lowest in infants (82.3, 65/79), but it was not statistically significant (X²=1.396, P<0.001).

Positive rates of different detection methods for M. pneumoniae

In these 830 children, positive rates of MP-IgM (PA), MP-IgM (GICT), MP-RNA (SAT) and 7 RNAs were respectively 40% (332/830), 26.7% (222/830), 17.6% (146/830) and 16.6% (138/830). MP-DNA (RT-PCR) results showed that the positive rate was 36.6% (304/830), of which the macrolide-resistant accounted for 86.2% (262/304) (Figure 1). Through 7 RNAs, we found the coinfection combination containing MP and RSV had the highest detection rate of 23.9% (33/138), followed by MP and Parainfluenza virus (21.7%, 30/138).

Diagnostic values of different methods with a real-time polymerase chain reaction as the standard

We compared diagnostic values of MP-IgM (PA), MP-IgM (GICT), MP-RNA (SAT) and 7 RNAs, considering the MP-DNA (RT-PCR) as the standard. As shown in Table 3, MP-IgM (PA) had good consistency (Kappa=0.526), sensitivity (74.0%) and Youden index (53.7%). And the specificity of MP-RNA (SAT) was the highest (97.5%), followed by 7 RNAs (89.2%). Moreover, comparing every two detection methods together, we found the combined MP-IgM (PA) and MP-RNA (SAT) was the best choice to detect M. pneumoniae, because its substantial Kappa value (Kappa=0.602) and its highest sensitivity (84.2%), specificity (78.7%) and Youden index (62.9%) were more than those of single method (Table 4).

In this study, we assessed the detection rate of different methods for M. pneumoniae pneumonia in different age groups and compared value of these diagnostic methods.

M. pneumoniae is a major cause of infectious diseases worldwide that affects different tissues and organs especially respiratory tracts of children in all age groups, and it has become the second leading pathogen after Streptococcus pneumoniae in children with community-acquired pneumonia.¹⁴ The occurrence of CAP caused by M. pneumoniae varies related to age. In an earlier research, M. pneumoniae pneumonia was reported uncommon in children under five years of age and higher incidence among school-age children.¹ However, M. pneumoniae infections may occur in persons from infancy up through old age.^15,16 From 2010 to 2012, 2638 children with pneumonia requiring hospitalization in three hospitals were conducted in the United States. Respiratory specimens were systematically collected and detected by using Real-Time PCR, and M. pneumoniae was found more common in children ≥5 years (19% vs. 3%).¹⁷ In accordance to our data, the detection rates of M. pneumoniae by different methods in three age groups were different, but the distributions seemed the same. School-age children and adolescents were the most common ages affected, and there was rarely a significant infection before 3 years old, which was consistent with the epidemiological characteristics of M. pneumoniae.

At present, there are many methods for laboratory diagnosis of M. pneumoniae infection, including culture, serological detection and molecular assay. Culture is often used for antimicrobial susceptibility testing or typing because of its high specificity. However, due to the complicated operation, prolonged turnaround time, and low sensitivity, culture is not recommended for routine testing.^18,19 Serological tests are the most widely used detections of M. pneumoniae in clinical practice currently, more sensitive than culture. Diagnostic sensitivity for serological tests of M. pneumoniae is decided by both the collection time of specimens and performance characteristics of the methods. In our study, the positive rate of MP-IgM (PA) was 40%, which higher than others, probably because the average time was 10 days before admission to our department. Although the MP-IgM is the earliest antibody produced after M. pneumoniae infection, it still takes a certain period of time. Usually it can be detected within about 1 week after infection, reaching a peak after 3 to 4 weeks, so false negative may appear in the early stage.²⁰ It was reported that the most accurate diagnostic of serology was obtained when paired sera collected at least 2 weeks apart were tested for both IgM and IgG at the same time and a 4-fold rise in titer could be showed.¹ But in pediatrics, it is simply impossible for repeated blood sampling in a short time. MP-IgM (GICT), a rapid test kit based on immuno-chromatography, is a relatively new serological test. One study from Wei Li found that the specificity and sensitivity of MP-IgM (GICT) were 100% and 97.4% respectively compared with real-time PCR,²¹ which was not in line with our result. It might be that the dilution of the sample only 100µl caused false negative. On account of the easy and rapid operation (15 minutes), MP-IgM (GICT) is suitable for the identification of M. pneumoniae infections in pediatric outpatient departments. However, MP-IgM (PA) is still recommended for inpatients in order to reduce false negative rate.

With the development of molecular assays, nucleic acid amplification technology (NAAT) has gradually become an important method for early rapid diagnosis of M. pneumoniae infection, which may help for early appropriate antibiotic therapy. Owing to the advantages of quick turnaround times, lower likelihood of contamination, higher sensitivity and specificity, not limited by the time and immune function, NAAT is of great significance in early diagnosis of M. pneumoniae. In addition, NAAT has various detection formats, which can provide quantitative data, detect antimicrobial resistance genes, and analyze genetic relatedness of the organisms.¹² However, there are a few limitations, such as contamination may result in false positives; it is difficult to obtain high quality samples; the time point for sampling effects results; and the PCR inhibitors can lead to false negative,²² which may be the reasons for the lower positive rate of NAAT in this study.

We found the rate of macrolide-resistant accounted for 86.2%. MRMP strains were increasing in many countries since the first case was reported in Japan in 2000,²³ with the long-term widespread use of macrolides. Macrolides act on the ribosomal 50S subunit to inhibit protein synthesis. Mutations in the V region of the MP 23S rRNA domain can cause a decrease in affinity between the drug and the ribosome, leading to drug resistance.²⁴ Common mutations including both positions 2063 and 2064 led to high-level resistance, whereas positions 2067 and 2617 were associated with low-level resistant.^25,26 In these 262 specimens, we only found a mutation at 2063. MRMP in the Beijing population reached a high rate of more than 90% from 2008 to 2012,²⁷ which was similar to our results.

MP-DNA detection by RT-PCR technology is to amplify gene fragments to diagnose pathogens, which has advantages in operation and sensitivity of detections.²⁸We compared diagnostic values of different methods for M. pneumoniae with RT-PCR as the standard. We found that MP-IgM (PA) had a high sensitivity of 74.0% and the highest Youden index and Kappa value, indicating that it was conducive to screening for MP infection. SAT is a recently developed method based on isothermal amplification of RNA,^29,30 which can be completed in about 3 hours. While in our study, the outcomes of SAT for M. pneumoniae, both MP-DNA and 7 RNAs, showed higher specificity but lower sensitivity. The specific RNA only exists in the proliferation stage of M. pneumoniae, so increases in RNA levels could reflect bacterial multiplication.³¹ Previous data showed that the SAT positivity rates were significantly higher in the untreated cases with MPP than in the macrolide-treated MPP cases.³² Therefore, one explanation for the “false-negative” SAT results was that long-term treatment might decrease the M. pneumoniae load. Our data showed that 78.3% of the cases received macrolide treatment before hospitalization. And another might relate to the possible poor operation skills and quality of the swab samples, leading to some samples being below the assay’s detection limit.^33,34 In addition, multiplex PCR assays could be used to detect M. pneumoniae and other respiratory pathogens, but monoplex assays had higher sensitivity and specificity versus multiplex assays,³⁵ which was confirmed by our results.

Each detection method of M. pneumoniae has its own advantages and disadvantages. Although molecular assays are superior in detecting M. pneumoniae because of rapid, sensitive and specific characteristics, they cannot take place of serology.³⁶ A research in China analyzed data from children hospitalized with MPP using IgM (PA) and RT-PCR.³⁷ The concordance was close to 90% for the two detection methods. However, 173 (7%) children with a positive PCR had a negative serological test. And there were only 72 (3%) IgM-positive children with PCR negative. Different detection methods can be used to not only improve diagnostic specificity and sensitivity but reduce false negative rate and false positive rate as well. It was indicated that no single test available could be reliable for the identification of M. pneumoniae infection and a combination of various methods turned out to be the most reliable approach.³⁸ A positive PCR without serological evidence of infection could mean that the specimen was collected too early in the course of M. pneumoniae infection for antibodies to develop. And PCR results could become negative after a period time of antibiotic treatment, meanwhile serological results would remain positive for a long time. Some reviews concluded that no single test could detect M. pneumoniae infection reliable but a combination of serological test and PCR might the most sensitive approach for early diagnosis in children.^12,39 In our research, two different M. pneumoniae detection methods were combined and evaluated by RT-PCR and the result showed that MP-IgM (PA) in combination with MP-RNA (SAT) could be used as good screening methods.

Our study had some limitations. First, this study was performed in a single center and included no data of clinical features, for there were no accurate ways to determine the specificity of these characteristics. Second, having no gold standard diagnostic assay, such as microbiological culture and four-fold rise of IgM and IgG in paired serum, made it hard to draw conclusions to confirm the most reliable and accurate method.

In conclusion, during the acute phases of M. pneumoniae pneumonia in children, detection of MP-IgM (PA) together with MP-RNA (SAT) proved to be a reliable and precise diagnosis. Pediatrician need to select appropriate detection methods based on the age of children and the onset of the disease, and synthesize various results to diagnosis M. pneumoniae infection.

MP: Mycoplasma pneumoniae; CAP: community-acquired pneumonia; PA: particle agglutination; GICT: immune colloidal gold technique; SAT: simultaneous amplification and testing; RT-PCR: real-time polymerase chain reaction; MRMP: macrolide-resistant mycoplasma pneumoniae; RSV: respiratory syncytial virus

Ethics approval and consent to participate

The study was approved by the Institutional Review Board and the Ethics Committee of Shanghai Children’s Medical Center (SCMCIRB-K2019060-1), and written informed consent was obtained from the parents of each patient.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used in the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

Not applicable.

Authors’ contributions

YY and QC initiated the study. MY-T and WD wrote the manuscript and analyzed the data. TX and YF-W analyzed the data and prepared Figs. JZ and LZ conducted statistical analyses and provided technical support. QC provided technical support. YY provided critical feedback. All authors read and approved the final manuscript.

Acknowledgments

We would like to thank the patients and their parents for the support and cooperation in this work.

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Table 1 General characteristics of the patients

Characteristic	Value for patients
Total	830
Age, No(%)
<3 years	421(50.7)
3-5 years	215(25.9)
6-15 years	194(23.4)
Sex, No(%)
Male	447(53.9)
Female	383(46.1)
Underlying diseases, No(%)
None	680(81.9)
Congenital heart disease	101(12.2)
Congenital biliary atresia	7(0.8)
Brain lesions	15(1.8)
Other diseases	27(3.3)
Need of oxygen
Yes	61(7.3)
No	769(92.7)
Prognosis after treat, No. (%)
Alive	825(99.4)
Death^a	5(0.6)

Four children died from congenital heart disease, and one died from primary immunodeficiency disease.

Table 2 Positive rates of methods in different age groups

Methods	<3years	3-5years	6-15years	X2	p
Methods	n=421(%)	n=215(%)	n=194(%)	X2	p
MP-IgM(PA)	91(21.6)	102(47.4)	139(71.6)	145.222	<0.001
MP-IgM(GICT)	66(15.7)	67(31.2)	89(45.9)	64.704	<0.001
MP-RNA(SAT)	36(8.6)	40(18.6)	70(36.1)	69.646	<0.001
7RNAs	71(16.9)	29(13.5)	38(19.6)	2.772	0.25
RT-PCR	79(18.8)	85(39.5)	140(72.2)	164.208	<0.001
Resistance	65(15.4)	74(34.4)	123(63.4)	142.516	<0.001

SAT: simultaneous amplification and testing

PA: particle agglutination

GICT: immune colloidal gold technique

RT-PCR: real-time polymerase chain reaction

Table 3 Diagnostic values of different methods with a real-time polymerase chain reaction as the standard

	RT-PCR		Kappa	Sensitivity	Specificity	Accuracy	Youden Index
	Positive	Negative
MP-IgM (PA)
Positive	225	107	0.526	74.0%	79.7%	77.6%	53.7%
Negative	79	419
MP-IgM (GICT)
Positive	151	71	0.384	49.7%	86.5%	73.0%	36.2%
Negative	153	455
MP-RNA (SAT)
Positive Negative	133 171	13 513	0.464	43.8%	97.5%	77.8%	41.2%
7 RNAs
Positive	81	57	0.179	26.6%	89.2%	66.3%	15.8%
Negative	223	469

SAT: simultaneous amplification and testing

PA: particle agglutination

GICT: immune colloidal gold technique

Table 4 Diagnostic values of combined different methods with a real-time polymerase chain reaction as the standard

	RT-PCR		Kappa	Sensitivity	Specificity	Accuracy	Youden Index
	Positive	Negative
MP-RNA (SAT)+MP-IgM (PA)
Positive	256	112	0.602	84.2%	78.7%	80.7%	62.9%
Negative	48	414
MP-RNA (SAT)+MP-IgM (GICT)
Positive	223	76	0.591	73.4%	85.6%	81.1%	59.0%
Negative	81	450
MP-IgM (GICT)+MP-IgM (PA)
Positive	237	123	0.525	78.0%	76.6%	77.2%	54.6%
Negative	67	403
7 RNAs+MP-IgM (PA)
Positive	250	154	0.419	82.2%	70.7%	74.9%	52.9%
Negative	54	372
7 RNAs+MP-IgM (GICT)
Positive	198	120	0.419	65.1%	77.2%	72.8%	42.3%
Negative	106	406

SAT: simultaneous amplification and testing

PA: particle agglutination

GICT: immune colloidal gold technique

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Comparison of different detection methods for Mycoplasma pneumoniae infection in children with community-acquired pneumonia

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