Rapid diagnosis of Mycoplasma pneumoniae in children with pneumonia by an immuno-chromatographic antigen assay.

Mycoplasma pneumoniae is a particularly important pathogen that causes community acquired pneumonia in children. In this study, a rapid test was developed to diagnose M. pneumoniae by using a colloidal gold-based immuno-chromatographic assay which targets a region of the P1 gene. 302 specimens were analyzed by the colloidal gold assay in parallel with real-time PCR. Interestingly, the colloidal gold assay allowed M. pneumoniae identification, with a detection limit of 1 × 10(3) copies/ml. 76 samples were found to be positive in both real-time PCR and the colloidal gold assay; two specimens positive in real-time PCR were negative in the rapid colloidal gold assay. The specificity and sensitivity of the colloidal gold assay were 100% and 97.4%, respectively. These findings indicate that the newly developed immuno-chromatographic antigen assay is a rapid, sensitive and specific method for identifying M. pneumoniae, with potential clinical application in the early diagnosis of Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (M. pneumoniae) is one of the main pathogens that cause community-acquired pneumonia (CAP) in children123. It can trigger a series of pathophysiologic responses, which may cause respiratory tract symptoms (paroxysmal irritating cough, sore throat, sticky sputum, and/or purulent sputum) and various complications such as bronchial asthma, acute respiratory distress syndrome, Guillain-Barre syndrome, polyarthritis, and coronary artery diseases456. At present, several methods are available for the diagnosis of M. pneumoniae infection, including culture, serological tests, and real time PCR techniques789. However, M. pneumoniae culture and serological tests are insensitive, time-consuming and cross-reactive; therefore, they are not appropriate for rapid and accurate detection of M. pneumoniae infection in clinical practice9. Recently, real-time PCR has been reported by many authors as a rapid, sensitive, and specific method, but it still requires at least 2–4 hours for DNA extraction and amplification10111213. In addition, real-time PCR may not discriminate live M. pneumoniae from dead ones9. In this study, we developed a rapid test kit based on immuno-chromatography by using a pair of monoclonal antibodies which target the conserved region of the P1 surface protein of M. pneumoniae. Then, a large sample size study was conducted to assess the clinical diagnostic value of the newly developed system, in comparison with that of a commercial real-time PCR assay.

Results

M. pneumoniae detection in the colloidal gold assay

As shown in Fig. 1, M. pneumoniae presence in a sample resulted in both the test and control lines being positive. A sample without M. pneumoniae displayed only a positive control line. To confirm the detection capacity of the colloidal gold assay, P1 genes of standard types I and II M. pneumoniae strains were tested. The results showed that both FH (type II M. pneumoniae) and M129 (type I M. pneumoniae) strain were positive in the colloidal gold assay (Fig. 2).

Figure 1

Colloidal gold plate setup (A) is representative of a negative sample, and (B) a positive sample).

Figure 2

FH and M129 test results in the colloidal gold assay.

Sensitivity and specificity of the colloidal gold assay for M. pneumoniae

To evaluate the specificity of the colloidal gold assay, 105 copies/ml of M. pneumoniae, Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, Klebsiella pneumoniae, Legionella pneumophila, Haemophilus influenzae, Pseudomonas aeruginosa, anaerobic bacteria, Chlamydia trachomatis, Chlamydia pneumoniae, adenovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, human cytomegalovirus, human metapneumovirus and enteroviruses were assessed in 3 independent experiments. Our results showed that M. pneumoniae was identified correctly by the colloidal gold assay with no cross-reactions found between M. pneumoniae and other pathogens (Data not shown).

To assess the sensitivity of the colloidal gold assay, standard M. pneumoniae was quantified by real-time PCR and submitted to serial 10-fold dilutions to obtain 101 to 107 copies/ml. Different concentrations of standard M. pneumoniae were tested by the colloidal gold assay. As shown in Fig. 3, M. pneumoniae with concentrations of 103–107copies/ml were positive in the colloidal gold assay (3 independent experiments). M. pneumoniae was not detected at 101–102 copies/ml.

Figure 3

Sensitivity of the colloidal gold assay.

(A) 107 copies/ml; (B) 106 copies/ml; (C) 105 copies/ml; (D) 104 copies/ml; (E) 103 copies/ml; (F) 102 copies/ml; (G) 101 copies/ml; (H) Negative control.

Clinical specimen data

A total of 78 throat swabs and 224 sputum specimens were collected from children with suspected M. pneumoniae infection. As shown in OTable 1, 78 (78/302, 25.8%) specimens were positive in real-time PCR. M. pneumoniae amounts ranged from 1 × 103 to 8.65 × 107 copies/ml. In the colloidal gold assay, 76 (76/302, 25.2%) samples were positive. The 76 specimens positive in the colloidal gold assay were also in real-time PCR; the corresponding patients were admitted to the hospital with a disease course of 5–10 days. There was a high statistical consistency (kappa value = 0.98, p = 0.000) between the colloidal gold assay and real-time PCR, indicating a high specificity for the newly developed method. Compared with real-time PCR, the specificity and sensitivity of the colloid gold assay were 100% and 97.4%, respectively. Only two samples were negative in the colloidal gold assay and positive in real-time PCR. Finally, M. pneumoniae DNA amounts in two samples were confirmed, with 1.3 × 103 and 2.2 × 103 copies/ml, respectively.

Table 1

Comparison of detection results between the colloidal gold assay and real-time PCR.

Colloidal gold assay	Real-time PCR		Total
	Positive	Negative
Positive	76	0	76
Negative	2	224	226
Total	78	224	302

Discussion

M. pneumoniae is a common pathogen of primary atypical pneumonia and other respiratory infectious diseases1415. In addition, it is one of the most important agents of acute respiratory infections in children between the ages of 5 and 15 years16. Our previous study revealed a M. pneumoniae pneumonia infection rate of 18.5% in children of Hangzhou (China)3. A rapid and accurate method for diagnosing M. pneumoniae pneumonia is needed. Real-time PCR and serological tests are currently used to diagnose M. pneumoniae clinically in China89. But time-consuming and complicated protocols restrict their clinical application. Recently, Miyashita et al. reported a diagnostic sensitivity of only 60% that of real-time PCR for a commercial rapid antigen kit (Asahi Kasei Pharma Co., Tokyo, Japan)20. In this study, we applied colloidal gold assay to detect M. pneumoniae by targeting the specific P1 antigen. P1 is one of the major surface proteins of M. pneumoniae and its gene is an attractive target in the clinical detection of M. pneumoniae by real-time PCR1017. This is the first study detecting P1 antigen to confirm M. pneumoniae infection in children with pneumonia. Clinical M. pneumoniae strains can be divided into two types (I and II) according to their P1 gene variants18. Colloidal gold assay has high capacity to detect both types of M. pneumoniae. M. pneumoniae detection could be completed in 15 minutes by using our method, while real-time PCR requires 2 to 4 hours, and culture usually takes 21 days19. Moreover, this method presents high sensitivity and specificity in detecting M. pneumoniae (no cross-reactions with other clinical pathogens), while serological tests often show lower specificity9. Of note, we found 103 copies/ml was the detection limit for this new method, while this value is 8.3 × 104 copies/ml for the Asahi company rapid antigen kit20.

In clinical practice, we used commercial real-time PCR assay as a control method, targeting the P1 gene of M. pneumoniae. We applied the newly developed colloidal gold assay to the 302 specimens from children with pneumonia. 76 (25.8%) specimens were positive for M. pneumoniae. When compared with real-time PCR, the specificity and sensitivity of the colloidal gold assay were 100% and 97.4%, respectively. The symptoms in all M. pneumoniae positive children were improved after treatment with Azithromycin. Two samples were positive in real time PCR but negative in the colloidal gold assay. Real-time PCR and clinical data were obtained from the two specimens, which both had 103 copies/ml. It should be noted that these 2 patients were treated with antibiotics for more than a week before visiting our hospital. We hypothesize that the two samples may have only contained low amounts of live M. pneumoniae, below the detection limit of the colloidal gold assay.

In conclusion, the colloidal gold assay is a rapid, sensitive, and specific method for the identification of M. pneumoniae. Most importantly, this method is easy to operate without any special instrument and may be suitable for bedside detection. These findings indicate that the newly developed colloidal gold assay would be an effective method for detecting M. pneumoniae in clinical practice.

Methods

M. pneumoniae strain and control strains

Standard M. pneumoniae FH (ATCC 15531) and M129 (ATCC 29342) strains were purchased from ATCC. Negative controls used in this study included Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, Klebsiella pneumoniae, Legionella pneumophila, Haemophilus influenzae, Pseudomonas aeruginosa, anaerobic bacteria, Chlamydia trachomatis, Chlamydia pneumoniae, adenovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, human cytomegalovirus, human metapneumovirus and enteroviruses. All negative controls were isolated from patients and conserved at the Department of clinical laboratory, Children’s Hospital of Zhejiang University School of Medicine.

Clinical specimens from children with pneumonia

From February to August 2014, 302 children were enrolled in this study. The inclusion criteria were: (1) age < 14 years; (2) patient visiting the Children’s Hospital of Zhejiang, University School of Medicine; (3) primary diagnosis as pneumonia according to known guidelines21. During six months, 302 specimens were collected, including 78 throat swabs and 224 sputum samples in our hospital. Each specimen was mixed with 1.5 ml saline and stored at −70 °C; 1 ml of the mixture was used for real-time PCR and 0.5 ml in the colloidal gold assay. The 302 patients (125 females and 177 males) were 3 months to 10 years old.

The study was performed in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Zhejiang University School of Medicine. All patients provided informed consent.

Preparation of the colloidal gold plate

As shown in Fig. 1, the colloidal gold plate contained three parts: 1) sample well; 2) reagent region; 3) chromatography region. The reagent region contained monoclonal antibody A (mouse) labeled with colloidal gold which targets the M. pneumoniae P1 antigen. The chromatography region included M. pneumoniae monoclonal antibody B (mouse) which is bound to the Test line position and mouse IgG polyclonal antibody bound to the Control line position in the chromatography. A sample with M. pneumoniae would result in monoclonal antibody A binding with M. pneumoniae and detected by monoclonal antibody B as well as mouse IgG polyclonal antibody (both of Test and Control lines positive). In a sample without M. pneumoniae, only monoclonal antibody A can be detected by IgG polyclonal antibody (Test line negative and Control line positive). The plate was manufactured by Genesis Biodetection & Biocontrol Ltd, Hangzhou, China.

Real-time PCR for M. pneumoniae detection

For real-time PCR, 1 ml of the mixture was transferred into a 1.5-ml microcentrifuge tube aseptically and centrifuged for 5 min at 12000 rpm/min. The cell pellets were resuspended in 50 μl lysis buffer (Da An Gene Co., Ltd., China); 4 μl lysate served as template in real-time PCR amplification based on the TaqMan probe PCR kit (Da An Gene Co., Ltd., China) as reported previously322. For each assay, negative and positive quality controls, and four positive quantity plasmid controls (104, 105, 106, and 107 copies/ml) were assessed. Ct values of the four quantity controls were then subjected to log-linear analysis to generate a standard curve used to determine the concentrations of clinical M. pneumoniae samples. Real-time PCR was carried out on an ABI 7500 instrument for 3 min at 95 °C, followed by 40 two-step cycles (15 s at 95 °C and 45 s at 55 °C).

Colloidal gold assay for M. pneumoniae detection

To perform the immune-chromatographic assay, 100 μl (about 3 drops) of the mixture was added into a sample well for 10–15 minutes. Samples with positive control and test lines were determined as M. pneumoniae positive; no Control line on the plate indicated an invalid test, and a second test plate was used till the result was either positive or negative.

Statistics

Statistical analysis was performed using the kappa test; statistical significance was calculated using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Additional Information

How to cite this article: Li, W. et al. Rapid diagnosis of Mycoplasma pneumoniae in children with pneumonia by an immuno-chromatographic antigen assay. Sci. Rep. 5, 15539; doi: 10.1038/srep15539 (2015).