OBJECTIVEThe P1 protein of Mycoplasma pneumoniae (MP) plays an important role in the pathogenesis of MP pneumonia. It mediates the attachment of the pathogen to host cells and elicits a strong humoral immune response during infection. In early studies, only two types of MP P1 genes were assumed to exist. Later, eight subtypes of MP P1 genes and some variations of P1 gene were reported. However, there are no related reports in China until now. This study aimed to understand epidemiology of MP subtype in Zhejiang province, China, as well as the relationship between MP subtype and clinical severity of MP pneumonia.

METHODClinical samples were collected by nasopharyngeal aspiration from children with MP pneumonia hospitalized in the Children's Hospital of Zhejiang University School of Medicine from February to December in 2009. P1 gene fragment was amplified by using PCR method (with primers of ADH1/ADH2 and ADH3/ADH4, respectively). Then ADH1/ADH2-generated fragments were digested with HaeIII, HpaII, Sau3A, and the ADH3/ADH4-generated fragments digested with HaeIII, Sau3A, HhaI, RsaI. The MP P1 subtypes were determined based on resulting fragments. Part of samples were selected for sequencing. The clinical data of different MP subtype pneumonia were compared.

RESULTA total of 300 hospitalized children with MP pneumonia were enrolled in this study. All the samples produced specific bands for MP P1 gene after PCR with primers of ADH1/ADH2 and ADH3/ADH4 respectively. By restrictive fragment length polymorphism analysis, 297 clinical specimens showed the characteristic band patterns for P1 type 1 identical to Mp129, and only 3 clinical specimens showed the characteristic band pattern for P1 type 2 identical to MP-FH. All P1 type 1 and P1 type 2 showed the same subtype bands respectively, as subtype 1b and 2a. After sequencing, one synonymous point mutation in P1 type 1 was identified relative to the MP129 P1 sequence at nucleotide position (nt) 208(G→A). Three cases with P1 type 2 MP pneumonia were found to have liver damage, and longer hospital stay and fever duration than P1 type 1, but no statistically significant difference was found.

CONCLUSIONClinical samples can be used directly for genotyping of MP. The dominating type of MP in Zhejiang Province was P1 type 1 subtype 1b. But whether there was any relationship between MP subtype and clinical severity remains to be clarified.

Adhesins, Bacterial ; genetics ; Child ; China ; DNA, Bacterial ; genetics ; Genotype ; Humans ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Nasopharynx ; microbiology ; Pneumonia, Mycoplasma ; microbiology ; Polymorphism, Restriction Fragment Length

OBJECTIVETo establish and evaluate a real-time PCR assay to detect Mycoplasma pneumoniae (M.pneumoniae) in clinical specimens.

METHODSBy analysing the whole p1 gene sequence of 60 M.pneumoniae clinical isolates in Beijing of China, an optimized real-time PCR assay (MpP1) using p1 gene conserved region was designed. The specificity and sensitivity of this assay were evaluated and compared with other two reported assays (RepMp1 and Mp181) using 40 positive and 100 negative clinical specimens.

RESULTSThe detection limit of the new assay was 8.1 fg (about 1∼3CFU) M.pneumoniae DNA. The sensitivity of MpP1, RepMp1, and Mp181 assays appeared to be 100%, 100%, and 85%, respectively.

CONCLUSIONMpP1 assay is suitable for the detection of M.pneumoniae in Chinese clinical specimens.

Genes, Bacterial ; Humans ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Sequence Analysis, DNA

OBJECTIVEMycoplasma pneumoniae (Mp) infection is one of major causes of community-acquired pneumonia. Isolation and culture of Mp are very difficult, fluorescent quantitative PCR is a new technique to detect Mp. The aim of this study was to explore the diagnostic value of fluorescent quantitation PCR for Mp infection.

METHODMp-DNA from the deep respiratory tract secretion of children suffering from pneumonia was tested by a fluorescent quantitative PCR. Totally 256 cases who were positive for Mp DNA were enrolled into this study, 164 (64.1%) were male, 92 (35.9%) were female; the age ranged from 9 days to 16 years. All the patients also had results of Mp-IgM test. These patients were divided into 2 groups according to the result of Mp-IgM detection, namely, Mp-IgM positive and negative groups. Area under the roc curve (Az) was used as the index to evaluate the diagnostic value of fluorescent quantitation PCR for Mp detection. The number of Mp-DNA copies, age and course of disease of the 2 groups were also compared.

RESULTS(1) Diagnostic accuracy of fluorescent quantitative PCR for detecting Mp infection was that Az = 0.641. (2) The number of copies of the cases in Mp-IgM positive group was 5.42 +/- 1.26 [log(Mp-DNA copy/ml)], while that of Mp-IgM-negative group was 4.87 +/- 1.29 [log(Mp-DNA copy/ml), t = 3.43, P < 0.05]. (3) The age of Mp-IgM positive group was dramatically younger than Mp-IgM negative group (P < 0.001).

CONCLUSIONThe diagnostic accuracy of fluorescent quantitative PCR for mycoplasma pneumoniae (Mp) infection is low; however for children whose immunologic systems are not fully developed, this technique has some diagnostic value, and higher number of Mp-DNA copies may support diagnosis of Mp infection.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Immunoglobulin M ; blood ; Infant ; Infant, Newborn ; Male ; Mycoplasma pneumoniae ; genetics ; immunology ; isolation & purification ; Pneumonia, Mycoplasma ; diagnosis ; Polymerase Chain Reaction ; methods

BACKGROUND: Differentiation of atypical pathogens is important for community-acquired pneumonia (CAP). In this study, we compared sputum and nasopharyngeal swabs (NPS) for use in detection of Mycoplasma pneumoniae (MP), Chlamydophila pneumoniae (CP), and Legionella pneumophila (LP), using Seeplex PneumoBacter ACE Detection Assay (PneumoBacter; Seegene). METHODS: Sputum and NPS specimens were collected from patients in 15 hospitals. DNA was extracted from sputum using QIAamp DNA Stool Mini Kit (Qiagen) and from NPS using easyMAG (bioMerieux). Both types of specimens were evaluated by multiplex PCR using PneumoBacter. To determine the diagnostic performance of this assay, sputum samples were also tested using BD ProbeTec ET Atypical Pneumonia Assay (APA; Becton Dickinson). RESULTS: Among 217 sputum and NPS, 20 (9.2%), 2 (0.9%), and 0 sputum were positive for MP, LP, and CP, respectively, whereas 8 (3.7%) NPS were positive for MP. The sputum APA test yielded 186, 206, and 204 interpretable results for MP, LP, and CP, respectively. Of these, 21 (11.3%) were positive for MP, 2 (1.0%) were positive for LP, and 0 samples were positive for CP. Compared to APA, the sensitivity and specificity of the sputum assay for MP were 95.2% and 100.0%, respectively, whereas for the NPS assay, these were 38.1% and 93.9%. Sputum testing was more sensitive than NPS testing (P=0.002). For LP and CP diagnosis, PneumoBacter and APA tests agreed 100%. CONCLUSIONS: Specimen type is crucial and sputum is preferred over NPS for simultaneous detection of MP, LP, and CP using multiplex PCR in CAP.
Chlamydophila Infections/diagnosis ; Chlamydophila pneumoniae/*genetics/isolation & purification ; Community-Acquired Infections/*diagnosis ; DNA, Bacterial/analysis/isolation & purification ; Humans ; Legionella pneumophila/*genetics/isolation & purification ; Legionnaires' Disease/diagnosis ; Multiplex Polymerase Chain Reaction ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Nasopharynx/*microbiology ; Pneumonia, Mycoplasma/diagnosis ; Reagent Kits, Diagnostic ; Sputum/*microbiology

OBJECTIVEMycoplasma pneumoniae (MP) is an important pathogen for community-acquired pneumonia in children. MP infection was considered to be self-limited, but many severe refractory MP pneumonia cases have been reported in recent years. The reason for variation in severity of MP pneumonia remains unclear. MP virulence including drug-resistance and host immunologic function are important influencing factors. The present study aimed to clarify relationship between local MP load and severity of MP pneumonia.

METHODMP DNA was quantitatively detected by fluorescent real-time PCR in bronchoalveolar lavage fluid (BALF) from 77 children with MP pneumonia. They were classified into groups of low MP load ( < 10(3)/ml, n = 14) , moderate MP load (10(3)-10(6)/ml, n = 22) and high MP load ( > 10(6)/ml, n = 41) . Clinical symptoms, main laboratory and imaging results of children among the three groups were compared.

RESULTWhen compared with low load group and moderate load group, high load group had longer fever duration (7 d, 10 d vs. 12 d) , longer time to normalization of temperature with macrolide administration (4 d, 8 d vs. 10 d) , more patients with high fever (50.0%, 68.2% vs. 87.8%) and longer duration of fever than 10 d (35.7%, 50.0% vs. 73.2%).Statistically significant difference existed in CRP among the three groups (1.0 mg/L, 11.5 mg/L, 34 mg/L). Large field of consolidation or atelectasis were found in 58.5% of high load patients, much higher than 22.7% in moderate load and 14.3% in low load patients. Bilateral or massive pleural effusion was not found in low load group, while in moderate load and high load group, they were 13.6% and 24.4%. However, no significant difference was found in symptoms and main laboratory and imaging results among different age groups in high load patients.

CONCLUSIONThere is a close relationship between MP load in BALF and clinical characteristics in children with MP pneumonia. Those with high MP load have a more severe process.

Adolescent ; Bacterial Load ; Bronchoalveolar Lavage Fluid ; microbiology ; Child ; Child, Preschool ; Colony Count, Microbial ; DNA, Bacterial ; genetics ; Female ; Humans ; Infant ; Male ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Pneumonia, Mycoplasma ; microbiology ; pathology ; Real-Time Polymerase Chain Reaction ; Severity of Illness Index

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged > or = 18 yr) who had an acute illness with a new cough and sputum (< or = 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and beta-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.
Anti-Bacterial Agents/therapeutic use ; Bordetella parapertussis/genetics/*isolation & purification ; Bordetella pertussis/genetics/*isolation & purification ; Bronchitis/drug therapy/*microbiology ; Chlamydophila pneumoniae/genetics/*isolation & purification ; Community-Acquired Infections/microbiology ; Female ; Humans ; Hypertension/complications ; Legionella pneumophila/genetics/*isolation & purification ; Male ; Middle Aged ; Mycoplasma pneumoniae/genetics/*isolation & purification ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Sputum/microbiology

We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene.
Anti-Bacterial Agents/pharmacology ; Child ; Child, Preschool ; Drug Resistance, Bacterial/drug effects ; Humans ; Macrolides/pharmacology ; Male ; Mutation ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Pneumonia, Mycoplasma/*diagnosis/microbiology/radiography ; RNA, Ribosomal, 23S/*analysis ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Siblings

Local epidemiologic data on the etiologies of patients hospitalized with community-acquired pneumonia (CAP) is needed to develop guidelines for clinical practice. This study was conducted prospectively to determine the proportion of atypical bacterial pathogens in adults patients hospitalized with CAP in Korea between October 2001 and December 2002. Microbiological diagnosis was determined by serology for antibodies to Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneu-mophila. Nucleic acid of M. pneumoniae and C. pneumoniae in respiratory samples and Legionella antigen in urine samples were detected. The study population consisted of 126 patients (71 males, 55 females), averaging 54.6 yr (SD+/-17.8), whose paired sera were available. An etiologic diagnosis for atypical pathogens was made in 18 patients (14.3%): C. pneumoniae 9 (7.1%), M. pneumoniae 8 (6.3%), and L. pneumophila 3 patients (2.4%). Streptococcus preumoniae and other typical pathogens were isolated from 36 patients (28.6%). Of 126 patients, 16 (12.7%) were admitted to intensive care unit and atypical pathogens were identified in 5 patients (31.3%). Initial clinical features of patients with pneumonia due to atypical, typical or undetermined pathogens were indistinguishable. We conclude that atypical pathogens should be seriously considered in hospitalized patients with CAP, when initiating empiric treatment in Korea.
RNA, Ribosomal, 16S/genetics ; Prospective Studies ; Polymerase Chain Reaction ; Pneumonia, Bacterial/blood/*microbiology/urine ; Mycoplasma pneumoniae/genetics/immunology/*isolation & purification ; Middle Aged ; Male ; Legionella pneumophila/genetics/immunology/*isolation & purification ; Korea ; Humans ; Hospitalization/statistics & numerical data ; Female ; Community-Acquired Infections/microbiology ; Chlamydophila pneumoniae/genetics/immunology/*isolation & purification ; Antigens, Bacterial/urine ; Antibodies, Bacterial/blood ; Aged ; Adult

OBJECTIVETo establish a quick method to detect drug resistance of Mycoplasma pneumoniae (MP) and study the condition of drug resistance in MP infection.

METHODSMP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability.

RESULTSOf the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064, the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10(2) ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test.

CONCLUSIONSThe gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.

Adolescent ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Female ; Genes, rRNA ; Humans ; Infant ; Male ; Microbial Sensitivity Tests ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Point Mutation ; Polymerase Chain Reaction ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 23S ; genetics

BACKGROUND: This study aimed to evaluate the prevalence of Mycoplasma pneumoniae in primary and tertiary care hospitals and its macrolide resistance rate. METHODS: Nasopharyngeal swabs were collected from 195 pediatric patients in primary and tertiary care hospitals from October to November 2010. The AccuPower MP real-time PCR kit (Bioneer, Korea) was used for the detection of M. pneumoniae. Direct amplicon sequencing was performed to detect point mutations conferring resistance to macrolides in the 23S rRNA gene. RESULTS: Among the 195 specimens, 17 (8.7%) were M. pneumoniae positive, and 3 of the strains (17.6%) obtained from these 17 specimens displayed the A2063G mutation in 23S rRNA. Three macrolide-resistant M. pneumoniae isolates were isolated from patients hospitalized at the primary care hospital. The positive rates of M. pneumoniae for the primary and tertiary care hospitals were 12.1% (15/124) and 2.8% (2/71), respectively (P=0.033). CONCLUSIONS: The positive rate of M. pneumoniae in the primary care hospital was higher than that in the tertiary care hospital. Simultaneous detection of M. pneumoniae and macrolide-resistant mutation genes in the 23S rRNA by real-time PCR is needed for rapid diagnosis and therapy of M. pneumoniae infections.
Anti-Bacterial Agents/*pharmacology ; Child, Preschool ; Drug Resistance, Bacterial/*drug effects ; Female ; Humans ; Infant ; Infant, Newborn ; Macrolides/*pharmacology ; Male ; Mycoplasma pneumoniae/genetics/*isolation & purification ; Nasopharynx/microbiology ; Pneumonia, Mycoplasma/epidemiology/microbiology ; Primary Health Care ; RNA, Ribosomal, 23S/analysis ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Tertiary Healthcare