Psittacosis is typically a mild febrile respiratory illness caused by infection with the bacterium Chlamydia psittaci and usually transmitted to humans by infected birds (1). On average, 11 psittacosis cases per year were reported in the United States during 2000-2017. During August-October 2018, the largest U.S. psittacosis outbreak in 30 years (82 cases identified*) occurred in two poultry slaughter plants, one each in Virginia and Georgia, that shared source farms (2). CDC used C. psittaci real-time polymerase chain reaction (PCR) to test 54 human specimens from this outbreak. This was the largest number of human specimens from a single outbreak ever tested for C. psittaci using real-time PCR, which is faster and more sensitive than commercially available serologic tests. This represented a rare opportunity to assess the utility of multiple specimen types for real-time PCR detection of C. psittaci. C. psittaci was detected more frequently in lower respiratory specimens (59% [10 of 17]) and stool (four of five) than in upper respiratory specimens (7% [two of 28]). Among six patients with sputum and nasopharyngeal swabs tested, C. psittaci was detected only in sputum in five patients. Cycle threshold (Ct) values suggested bacterial load was higher in lower respiratory specimens than in nasopharyngeal swabs. These findings support prioritizing lower respiratory specimens for real-time PCR detection of C. psittaci. Stool specimens might also have utility for diagnosis of psittacosis.

New diagnostic platforms often use naso- or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multi-pathogen testing to high-quality sputum specimens to determine if more pathogens could be identified relative to NP/OP swabs. Children (<18 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 ≤10 epithelial cells/low power field [lpf] and ≥25 white blood cells/lpf or a q-score definition of 2+) were tested by TaqMan Array Card (TAC), a multi-pathogen real-time polymerase chain reaction (PCR) detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% v. 68%, P<0.0001) and adults (88% v. 61%; P<0.0001); for each pathogen targeted, crossing threshold (Ct) values were earlier in sputum. Both bacterial (361 vs. 294) and viral detections (245 vs. 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.

On June 20, 2014, a Nebraska long-term care facility notified the East Central District Health Department (ECDHD) and Nebraska Department of Health and Human Services (NDHHS) of an outbreak of respiratory illness characterized by cough and fever in 22 residents and resulting in four deaths during the preceding 2 weeks. To determine the etiologic agent, identify additional cases, and implement control measures, Nebraska and CDC investigators evaluated the facility's infection prevention measures and collected nasopharyngeal (NP) and oropharyngeal (OP) swabs or autopsy specimens from patients for real-time polymerase chain reaction (PCR) testing at CDC. The facility was closed to new admissions until 1 month after the last case, droplet precautions were implemented, ill residents were isolated, and group activities were canceled. During the outbreak, a total of 55 persons experienced illnesses that met the case definition; 12 were hospitalized, and seven died. PCR detected Mycoplasma pneumoniae DNA in 40% of specimens. M. pneumoniae should be considered a possible cause of respiratory illness outbreaks in long-term care facilities. Morbidity and mortality from respiratory disease outbreaks at long-term care facilities might be minimized if facilities monitor for respiratory disease clusters, report outbreaks promptly, prioritize diagnostic testing in outbreak situations, and implement timely and strict infection control measures to halt transmission.

Members of the Gram-negative genus Legionella are typically found in freshwater environments, with the exception of L. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires' disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genus Legionella are capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases' homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species of Legionella represented by 87 isolates.

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.

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.

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.

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 HRM genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.

Four nucleic acid extraction procedures (2 automated and 2 manual) were compared for their efficiency at isolating Mycoplasma pneumoniae DNA. Oropharyngeal swabs from healthy volunteers were spiked with varying amounts of M. pneumoniae, extracted, and tested using real-time polymerase chain reaction. Our data indicate that both automated extraction methods consistently outperform the manual procedures.

Chlamydophila pneumoniae is an atypical bacterial respiratory pathogen that is responsible for approximately 3-10% of community-acquired pneumonia cases. We report the evaluation of two distinct real-time PCR assays for rapid and specific detection of C. pneumoniae. We tested 401 clinical specimens, finding 5.7% positive, and confirmed a localized outbreak.