Use of Real-Time PCR for Chlamydia psittaci Detection in Human Specimens During an Outbreak of Psittacosis - Georgia and Virginia, 2018.

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.

Improved Detection of Respiratory Pathogens by Use of High-Quality Sputum with TaqMan Array Card Technology.

New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired 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 (<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 quality score [q-score] definition of 2+) 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 ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold (CT) 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.

Mycoplasma pneumoniae outbreak in a long-term care facility--Nebraska, 2014.

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.

The multivariate detection limit for Mycoplasma pneumoniae as determined by nanorod array-surface enhanced Raman spectroscopy and comparison with limit of detection by qPCR.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for up to 20% of community-acquired pneumonia. At present, the standard for detection and genotyping is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity but lacks standardization and has limited practicality for widespread, point-of-care use. We previously described a Ag nanorod array-surface enhanced Raman spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae in simulated and true clinical throat swab samples with statistically significant specificity and sensitivity. We report here that differences in sample preparation influence the integrity of mycoplasma cells for NA-SERS analysis, which in turn impacts the resulting spectra. We have established a multivariate detection limit (MDL) using NA-SERS for M. pneumoniae intact-cell sample preparations. Using an adaptation of International Union of Pure and Applied Chemistry (IUPAC)-recommended methods for analyzing multivariate data sets, we found that qPCR had roughly 10× better detection limits than NA-SERS when expressed in CFU ml(-1) and DNA concentration (fg). However, the NA-SERS MDL for intact M. pneumoniae was 5.3 ± 1.0 genome equivalents (cells per µl). By comparison, qPCR of a parallel set of samples yielded a limit of detection of 2.5 ± 0.25 cells per µl. Therefore, for certain standard metrics NA-SERS provides a multivariate detection limit for M. pneumoniae that is essentially identical to that determined via qPCR.

Comparison of real-time PCR and a microimmunofluorescence serological assay for detection of chlamydophila pneumoniae infection in an outbreak investigation.

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.

Comparison of laboratory diagnostic procedures for detection of Mycoplasma pneumoniae in community outbreaks.

BACKGROUND: Mycoplasma pneumoniae continues to be a significant cause of community-acquired pneumonia (CAP). A more definitive methodology for reliable detection of M. pneumoniae is needed to identify outbreaks and to prevent potentially fatal extrapulmonary complications. METHODS: We analyzed 2 outbreaks of CAP due to M. pneumoniae. Nasopharyngeal and/or oropharyngeal swab specimens and serum samples were obtained from persons with clinically defined cases, household contacts, and asymptomatic individuals. Real-time polymerase chain reaction (PCR) for M. pneumoniae was performed on all swab specimens, and the diagnostic utility was compared with that of 2 commercially available serologic test kits. RESULTS: For cases, 21% yielded positive results with real-time PCR, whereas 81% and 54% yielded positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M serologic tests, respectively. For noncases, 1.8% yielded positive results with real-time PCR, whereas 63% and 79% yielded serologically positive results with the immunoglobulin M and immunoglobulin G/immunoglobulin M kits, respectively. The sensitivity of real-time PCR decreased as the duration between symptom onset and sample collection increased, with a peak sensitivity of 48% at 0-21 days. A specificity of 43% for the immunoglobulin M antibody detection assay was observed for persons aged 10-18 years, but the sensitivity increased to 82% for persons aged 19 years. DISCUSSION: 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.

Identification of P1 variants of Mycoplasma pneumoniae by use of high-resolution melt analysis.

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.

Evaluation of two real-time PCR chemistries for the detection of Chlamydophila pneumoniae in clinical specimens.

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.

Evaluation of three real-time PCR assays for detection of Mycoplasma pneumoniae in an outbreak investigation.

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.

Survey of legionella species found in thai soil.

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.

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay.

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.

Comparison of nucleic acid extraction methods for the detection of Mycoplasma pneumoniae.

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.

Community outbreak of Mycoplasma pneumoniae infection: school-based cluster of neurologic disease associated with household transmission of respiratory illness.

BACKGROUND: We investigated an outbreak of severe neurologic disease and pneumonia that occurred among students at 4 schools in Rhode Island. METHODS: We identified cases of encephalitis, encephalomyelitis, and pneumonia that occurred among schoolchildren from 1 September 2006 through 9 February 2007, and we performed serologic tests, polymerase chain reaction (PCR) analysis, and culture for the detection of multiple pathogens in oropharyngeal and nasopharyngeal specimens. Students with positive results of M. pneumoniae IgM serologic testing and no alternative diagnosis were considered to be infected with M. pneumoniae. At school A, we used questionnaires to identify students and their household contacts who made visits to physicians for pneumonia and cough. We compared observed and expected rates of pneumonia. RESULTS: Rates of pneumonia among elementary students (122 cases/1000 student-years) were > 5-fold higher than expected. Three students had encephalitis or encephalomyelitis, and 76 had pneumonia. Of these 2 groups of students, 2 (66%) and 57 students (75%), respectively, had M. pneumoniae infection. M. pneumoniae was detected by PCR in 10 students with pneumonia; 5 of these specimens were cultured, and M. pneumoniae was isolated in 4. Of 202 households of students attending school A, 20 (10%) accounted for 61% of visits to physicians for pneumonia or cough. Of 19 household contacts of students with pneumonia, 8 (42%) developed pneumonia and 6 (32%) reported visits for cough. CONCLUSIONS: 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.