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 × 103 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.

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 × 10 3 to 8.65 × 10 7 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 × 10 3 and 2.2 × 10 3 copies/ml, respectively.   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 PCR 10,17 . 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 variants 18 . 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 days 19 . 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 specificity 9 . Of note, we found 10 3 copies/ml was the detection limit for this new method, while this value is 8.3 × 10 4 copies/ml for the Asahi company rapid antigen kit 20 .
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 10 3 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. 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 guidelines 21 . 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.

M. pneumoniae strain and control strains. Standard
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 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 μ l lysis buffer (Da An Gene Co., Ltd., China); 4 μ l lysate served as template in real-time PCR amplification based on the TaqMan probe PCR kit (Da An Gene Co., Ltd., China) as reported previously 3,22 . For each assay, negative and positive quality controls, and four positive quantity plasmid controls (10 4 , 10 5 , 10 6 , and 10 7 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 μ l (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).