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Mycoplasma pneumoniae is a significant cause of respiratory illness worldwide. Despite a minimal and highly conserved genome, genetic diversity within the species may impact disease. We performed whole genome sequencing (WGS) analysis of 107 M. pneumoniae isolates, including 67 newly sequenced using the Pacific BioSciences RS II and/or Illumina MiSeq sequencing platforms. Comparative genomic analysis of 107 genomes revealed >3,000 single nucleotide polymorphisms (SNPs) in total, including 520 type-specific SNPs. Population structure analysis supported the existence of six distinct subgroups, three within each type. We developed a predictive model to classify an isolate based on whole genome SNPs called against the reference genome into the identified subtypes, obviating the need for genome assembly. This study is the most comprehensive WGS analysis for M. pneumoniae to date, underscoring the power of combining complementary sequencing technologies to overcome difficult-to-sequence regions and highlighting potential differential genomic signatures in M. pneumoniae.

After returning from Europe to the United States, on March 1, 2020, a symptomatic teacher received positive test results for severe acute respiratory syndrome coronavirus 2. Of the 21 students exposed to the teacher in the classroom, serologic results suggested past infection for 2. Classroom contact may result in virus transmission.

Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.

[This corrects the article DOI: 10.1371/journal.pone.0174701.].

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.

Background : Identifying lung pathogens and acute spikes in lung counts remain a challenge in the treatment of patients with cystic fibrosis (CF). Bacteria from the deep lung may be sampled from aerosols produced during coughing. Methods : A new device was used to collect and measure bacteria levels from cough aerosols of patients with CF. Sputum and oral specimens were also collected and measured for comparison. Pseudomonas aeruginosa , Staphylococcus aureus , Klebsiella pneumoniae , and Streptococcus mitis were detected in specimens using Real-Time Polymerase Chain Reaction (RT-PCR) molecular assays. Results : Twenty adult patients with CF and 10 healthy controls participated. CF related bacteria (CFRB) were detected in 13/20 (65%) cough specimens versus 15/15 (100%) sputum specimens. Commensal S. mitis was present in 0/17 (0%, p=0.0002) cough specimens and 13/14 (93%) sputum samples. In normal controls, no bacteria were collected in cough specimens but 4/10 (40%) oral specimens were positive for CFRB. Conclusions : Non-invasive cough aerosol collection may detect lower respiratory pathogens in CF patients, with similar specificity and sensitivity to rates detected by BAL, without contamination by oral CFRB or commensal bacteria.

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.

ABSTRACT Mycoplasma pneumoniae type 2 strain FH was previously sequenced with Illumina (FH-Illumina) and 454 (FH-454) technologies according to Xiao et al. (2015) and Krishnakumar et al. (2010). Comparative analyses revealed differences in genomic content between these sequences, including a 6-kb region absent from the FH-454 submission. Here, we present a complete genome sequence of FH sequenced with the Pacific Biosciences RSII platform.

On October 28, 2011, the West Virginia Department of Health and Human Resources notified CDC of an increase in pneumonia cases among school-aged children in two rural counties. Mycoplasma pneumoniae was the suspected cause, based on epidemiology, clinical presentation, and testing of specimens sent to CDC. Three of six nasopharyngeal swabs were positive for M. pneumoniae in testing by quantitative real-time polymerase chain reaction (qPCR). The West Virginia Department of Health and Human Resources and CDC conducted an outbreak investigation to confirm the etiology of the outbreak, establish active case surveillance, and provide recommendations for treatment and containment. The investigation confirmed M. pneumoniae as the cause and identified 125 cases, including two caused by macrolide-resistant isolates. The outbreak was contained with public health interventions that included communicating to the public the importance of respiratory hygiene, providing hand sanitizer in schools, and informing health-care providers about macrolide resistance; antibiotic prophylaxis was not used. Despite the large number of cases and macrolide-resistant strains, no severe extrapulmonary manifestations (e.g., erythema multiforme) were reported.