Mycoplasma pneumoniae is a leading cause of respiratory infections, including community-acquired pneumonia (CAP). Currently, pathogen-specific testing is not routinely performed in the primary care setting, and the United States lacks a systematic surveillance program for M. pneumoniae. Documentation of individual cases and clusters typically occurs only when severe illness and/or failure to improve with empirical antibiotic therapy is observed. Outbreaks, some lasting for extended periods and involving a large number of cases, occur regularly. However, many more likely go unrecognized due to the lack of diagnostic testing and structured reporting. We reviewed data from 17 investigations of cases, small clusters, and outbreaks of M. pneumoniae M ycoplasma pneumoniae is a common respiratory pathogen in adults and children worldwide and is a leading cause of community-acquired pneumonia (CAP). The majority of infections are self-limiting, and empirical treatment without pathogen-specific diagnostic testing is common in the primary care setting. In fact, the current treatment guidelines do not recommend diagnostic testing for suspected CAP of atypical bacterial etiology in adults in the outpatient setting unless a change in the patient treatment regimen is anticipated (1). The recommendation to perform diagnostic testing for M. pneumoniae infection in children is classified as a weak recommendation and likely not routinely performed, in part due to a lack of available diagnostic tests in the primary care setting and at state and local public health laboratories (2). The estimates of the actual number of cases occurring annually are inexact due to the lack of systematic surveillance and reporting. Although historically noted to occur in 3-to 7-year cycles, outbreaks of M. pneumoniae are common and may last for several months as a result of the long incubation period and prolonged carriage after resolution of symptoms (3-5).Excessive or inappropriate antibiotic use provides selective pressure for the development of antimicrobial resistance. In M. pneumoniae infections, macrolide resistance is an emerging threat worldwide, and in some parts of the world, Ͼ90% of M. pneumoniae infections are caused by resistant strains (6, 7). In the United States, the incidence of infection with macrolide-resistant M. pneumoniae strains is not well defined, although the trait has been consistently reported over the past decade (8-10). Macrolide-resistant M. pneumoniae infection has been associated with increased febrile period, increased duration of persistent cough, and extended antibiotic therapy compared to macrolide-sensitive strains (11-16).Methods for molecular typing of M. pneumoniae strains have been developed, including discrimination of two major types and variants based on the sequence of the P1 adhesion molecule gene and identification of multiple types using multilocus variablenumber tandem-repeat analysis (MLVA) to examine four or five polymorphic loci. Despite development of these sophisticated typing methods, character...

In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n ‫؍‬ 4) number of M. pneumoniae-positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations. Mycoplasma pneumoniae is one of the most common bacterial etiologies of community-acquired pneumonia (CAP), representing 15 to 20% of cases in some studies (28). It is also a significant cause of community-wide outbreaks which are reported to occur in 3-to 7-year intervals with various incidence rates (10,12,28). Upper and lower respiratory tract symptoms are often mild and self-limiting; however, occasional extrapulmonary complications may develop, sometimes resulting in death (5,7,23).Strain subtyping is an important epidemiological tool for surveillance and outbreak investigations. Until recently, molecular typing of M. pneumoniae had been hindered by its minimal and highly uniform genome. Molecular typing was often restricted to a single gene encoding the P1 protein. Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4, 18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6, 21, 22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al. (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types. This procedure takes advantage of the variation in the copy numbers of tandemly repeated sequences within five different loci within the genome and has proven to be highly discriminatory (...

A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

BACKGROUND: Stevens-Johnson syndrome (SJS) is an uncommon, sporadic disease and outbreaks are rare. In November 2013, an outbreak of SJS was identified at Children's Hospital Colorado.METHODS: Outbreak cases were children aged 5-21 with a discharge diagnosis of SJS admitted from September 1 to November 30, 2013. Medical charts were reviewed using standardized data collection forms. Respiratory specimens were tested for viruses and Mycoplasma pneumoniae (Mp) by polymerase chain reaction (PCR). We conducted a separate 4-year retrospective case-control study comparing hospitalized SJS cases with and without evidence of Mp infection.RESULTS: During the outbreak, 8 children met SJS criteria. Median age was 11.5 years (range 8-16 years); 5 (63%) were boys and 5 (63%) were Mp-PCR-positive. Of the 5 PCR-positive children, none had preceding medication exposure, and all had radiographic pneumonia. All outbreak Mp isolates were macrolide susceptible. The retrospective case-control analysis showed that Mp-associated SJS episodes (n = 17) were more likely to have pneumonia (odds ratio [OR] 10.0, confidence interval [CI] 1.3-5.1), preceding respiratory symptoms (OR 30.0, CI 1.6-72.6), an erythrocyte sedimentation rate $35 mg/dL (OR 22.8, CI 2.1-244.9), and #3 affected skin sites (OR 4.5, CI 1.2-17.4) than non-Mp-associated SJS episodes (n = 23). CONCLUSIONS:We report the largest outbreak of SJS in children, which was also predominately associated with Mp infection. Mp-associated SJS was associated with a distinct clinical presentation that included less extensive skin disease, an elevated erythrocyte sedimentation rate, and evidence of a preceding respiratory infection.WHAT'S KNOWN ON THIS SUBJECT: StevensJohnson syndrome (SJS) is a rare and severe immunologic phenomenon characterized by rash and mucous membrane disease. SJS may be triggered by medications and, less commonly, by infections such as Mycoplasma pneumoniae (Mp). Outbreaks of SJS are exceedingly rare. WHAT THIS STUDY ADDS:We describe the largest SJS outbreak reported in children, which was also Mp-associated. In the first case-control study of this disease, we identify predictors of Mp-associated SJS versus non-Mp-associated SJS, including fewer skin lesions, pneumonia, and elevated erythrocyte sedimentation rate.

An outbreak at a university in Georgia was identified after 83 cases of probable pneumonia were reported among students. Respiratory specimens were obtained from 21 students for the outbreak investigation. The TaqMan array card (TAC), a quantitative PCR (qPCR)-based multipathogen detection technology, was used to initially identify Mycoplasma pneumoniae as the causative agent in this outbreak. TAC demonstrated 100% diagnostic specificity and sensitivity compared to those of the multiplex qPCR assay for this agent. All M. pneumoniae specimens (n ‫؍‬ 12) and isolates (n ‫؍‬ 10) were found through genetic analysis to be susceptible to macrolide antibiotics. The strain diversity of M. pneumoniae associated with this outbreak setting was identified using a variety of molecular typing procedures, resulting in two P1 genotypes (types 1 [60%] and 2 [40%]) and seven different multilocus variable-number tandem-repeat analysis (MLVA) profiles. Continued molecular typing of this organism, particularly during outbreaks, may enhance the current understanding of the epidemiology of M. pneumoniae and may ultimately lead to a more effective public health response.

We report molecular characteristics of M. pneumoniae in respiratory specimens from children and adults hospitalized with CAP. The P1 type 1 genotype and MLVA type 4/5/7/2 predominated, but proportions of types differed between children and adults. Macrolide resistance was rare.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

These results demonstrate that the variability of secretion of cytokines and expression of CCRs detected in DCs stimulated with different serotypes of A. actinomycetemcomitans or P. gingivalis is TLR2 or TLR4 dependent, respectively.