We compared the performances of three recently optimized real-time PCR assays derived from distinct genomic regions of Mycoplasma pneumoniae during an outbreak. Comprehensive evaluation established that a newly described toxin gene represents a superior target for detecting M. pneumoniae DNA in clinical specimens, although use of multiple targets may increase testing confidence.Mycoplasma pneumoniae accounts for approximately 15% to 20% of all community-acquired pneumonia cases and is a common cause of outbreaks (10,16,18). Outbreaks have been reported to occur in 3-to 7-year intervals, and although all age groups are susceptible, incidence rates vary with age and may occur more frequently in certain settings (10,16,17). M. pneumoniae infection spreads efficiently within households and close living quarters, with incubation periods as long as 3 weeks (17). The insidious nature of this infection and its protracted disease course make this agent a predominant cause of "walking pneumonia" that can persist within the population and cause community or institutional outbreaks. Upper-and lowerrespiratory-tract symptoms are often mild, resulting in tracheobronchitis, headache, and cough. Occasionally, severe cases with extrapulmonary involvement can result in hospitalization and death due to neurological disease, such as encephalitis (1,2,4,15,17). The high rate of morbidity and the occasional mortality reinforces the need for timely diagnosis for administering proper antibiotic treatment (7, 9).Conventional tests for detecting M. pneumoniae are fraught with limitations (3). M. pneumoniae culture can often take several weeks, requires special media and expertise, and is insensitive and prone to contaminants and inhibitors. Serological assays such as complement fixation and commercially available immunoglobulin detection kits are by nature retrospective, requiring paired serum samples from both acute and convalescent phases, and provide questionable specificity and sensitivity results. In sum, these approaches are impractical for a rapid diagnosis. A variety of nucleic acid-based tests based upon PCR have been developed for the rapid and sensitive detection of M. pneumoniae (5,11,13,14,16). The range of variables within each PCR study (specimen type, nucleic acid extraction and amplification procedures, target selection, definitions used in calculating data, etc.) makes it difficult to compare results and draw a single, comprehensive approach for reliable detection.Recent community outbreaks of M. pneumoniae infection underscore a need among public health departments and local hospitals for a rapid and reliable diagnostic assay (1,10,12,17,18). Moreover, this test should be highly specific and sensitive and should be evaluated in an outbreak setting. The aim of the current study was to evaluate the use of three recently optimized real-time PCR assays for the detection of M. pneumoniae in respiratory samples from a recent outbreak. To our knowledge, this is the first prospective and comparative study of real-time PCR targ...

Thorough data analysis indicated that no single available test was reliable for the identification of an outbreak of CAP due to M. pneumoniae. A combination of testing methodologies proved to be the most reliable approach for identification of outbreaks of CAP due to M. pneumoniae, especially in the absence of other suspected respiratory pathogens.

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.

New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with communityacquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (Ͻ18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as Յ10 epithelial cells/lowpower field [lpf] and Ն25 white blood cells/lpf or a quality score [q-score] definition of 2ϩ) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had Ն1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P Ͻ 0.0001) and adults (88% versus 61%; P Ͻ 0.0001); for each pathogen targeted, crossing threshold (C T ) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.KEYWORDS community-acquired pneumonia, pneumonia, multipathogen, diagnostics, TaqMan array card, NP/OP, TAC, sputum A cute respiratory infections (ARI), especially lower respiratory tract (LRT) infections, including community-acquired pneumonia (CAP), are a significant cause of morbidity and mortality globally (1-3). Despite advances in diagnostic testing beyond culture-based methods, including molecular and antigen-based approaches, pneumonia etiology often remains undetermined even when systematic and comprehensive specimen collection and methods are employed, particularly in adults (1, 2); one reason for this is that specimens directly from the lungs are often not available. Upper respiratory tract specimens, such as nasopharyngeal and oropharyngeal (NP/OP) swabs, are often collected for molecular testing of respiratory pathogens due to the ease of collection. Some studies suggest that LRT specimens, such as sputum, endotracheal aspirates (ETA), and bronchoalveolar lavage (BAL) fluids, have improved sensitivity compared with NP/OP swabs; while issues of specificity needed to inform clinician

M. pneumoniae caused a community-wide outbreak of cough illness and pneumonia and was associated with the development of life-threatening neurologic disease. Although M. pneumoniae was detected in schools, its transmission in households amplified the outbreak. Interrupting household transmission should be a priority during future outbreaks.

Mycoplasma pneumoniae is an important respiratory pathogen, accounting for up to 25% of community-acquired pneumonia, and is a common cause of hospitalized pneumonia in otherwise healthy adults and children. Mycoplasma pneumoniae isolates can be classified into two main genomic groups (type 1 and type 2) based on sequence variation within the gene encoding the major adhesion molecule P1. Although numerous publications have described real-time PCR assays for the detection of M. pneumoniae, none has been able to discriminate the two genomic types. Here, a real-time PCR assay that can distinguish each type of M. pneumoniae utilizing high-resolution melt-curve analysis is reported. Using this method, 102 isolates obtained from patients from 1965 to the present, including those from recent outbreaks, were typed along with reference strains M129 (type 1) and FH (type 2). The results show that 55 isolates (54%) can be classified as type 1 and 47 isolates (46%) as type 2, and 100% correlation was demonstrated when compared with a standard PCR-restriction fragment length polymorphism typing procedure. Typing of isolates obtained from recent outbreaks in the USA has revealed the presence of both types. This assay provides a rapid, reliable and convenient method for typing M. pneumoniae isolates and may be useful for surveillance purposes and epidemiological investigations, and may provide insight into the biology of M. pneumoniae distribution within populations.

We assessed the performance of a recently validated real-time PCR assay and a commercially available microimmunofluorescence serologic test for the detection of Chlamydophila pneumoniae infection during an outbreak. Evaluation of specimens from 137 individuals suggests that real-time PCR holds greater utility as a diagnostic tool for early C. pneumoniae detection.

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia. Although two genetically distinct types of M. pneumoniae are known, variants of each also exist. We used a real-time PCR high-resolution melt genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.Mycoplasma pneumoniae is a pervasive pathogen that accounts for up to 20% of all community-acquired pneumonias (14,15). Although its genome is highly homogeneous between strains, the P1 gene, which encodes an immunogenic adhesion protein, serves as a target to categorize isolates into either a type 1 or a type 2 genetic group (3). Numerous reports of genetic variants of each type have also been described (2-4, 6, 8). Spuesens et al. recently showed that intragenomic homologous DNA recombination occurs within the RepMP2/3 and RepMP4 regions of the P1 gene (MPN141) of M. pneumoniae and strain differences can be attributed to variation within RepMP elements (13). Dumke et al. also reported that M. pneumoniae variants differ only within the RepMP2/3 element (5). The clinical relevance of these findings is unclear, but epidemiological evidence of temporally based type-specific immunity in the population suggests these variations impart an advantage to the organism (6, 11). Moreover, the well-documented 3-to 7-year cyclic patterns of population-based, typespecific outbreaks seem to support this hypothesis (7,9,11).Historically, typing schemes were based upon restriction fragment length polymorphism (RFLP) analysis of digested PCR products of the P1 gene; however, Degrange et al. recently described a multiple-locus variable-number tandem-repeat analysis assay that was able to sort 265 strains into 26 distinct groups (1). This assay requires amplification of five loci followed by capillary electrophoresis and analysis but provides a greater level of genomic resolution. We recently reported the development of a real-time PCR assay that is able to rapidly distinguish M. pneumoniae isolates into type 1 or type 2 categories (12). We used this technique to classify 102 isolates using real-time PCR followed by high-resolution melt (HRM) analysis of a variable region partially spanning the RepMP2/3 element of the P1 gene (10, 12). The current study reports the use of this assay to identify variants of each group based upon intratype sequence deviations. Through the analysis of distinct HRM profiles from clinical isolates obtained by the CDC Respiratory Diseases Branch, we have been able to identify variants of each type. In this report, we describe the identification, sequence analysis, and unique HRM profiles of three diverse P1 variants (isolates 3, 684, and 549) and demonstrate the utility of this assay for detecting M. pneumoniae variants along with prototypical type 1 and 2 strains.M. pneumoniae culture and isolation were performed as previously described (16). M129 (ATCC 29342) and FH (ATCC 15531) were used as the reference strains for type 1 and type 2, respectively. Nuclei...