Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

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.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

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.

Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.

During 2012-2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7-28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1-138.2) among M. pneumonia-positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%-98.5%) in SRI patients and 80.7% (95% CI 16.7%-95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2.

Mycoplasma hominis Infections Transmitted Through Amniotic Tissue Product.

Background: Mycoplasma hominis is a commensal genitourinary tract organism that can cause infections outside the genitourinary tract. We investigated a cluster of M. hominis surgical site infections in patients who underwent spine surgery, all associated with amniotic tissue linked to a common donor. Methods: Laboratory tests of tissue product from the donor, including culture, quantitative real-time polymerase chain reaction (qPCR), and whole-genome sequencing were performed. Use of this amniotic tissue product was reviewed. A multistate investigation to identify additional cases and locate any unused products was conducted. Results: Twenty-seven tissue product vials from a donor were distributed to facilities in 7 states; at least 20 vials from this donor were used in 14 patients. Of these, 4 of 14 (29%) developed surgical site infections, including 2 M. hominis infections. Mycoplasma hominis was detected by culture and qPCR in 2 unused vials from the donor. Sequencing indicated >99% similarity between patient and unopened vial isolates. For 5 of 27 (19%) vials, the final disposition could not be confirmed. Conclusions: Mycoplasma hominis was transmitted through amniotic tissue from a single donor to 2 recipients. Current routine donor screening and product testing does not detect all potential pathogens. Clinicians should be aware that M. hominis can cause surgical site infections, and may not be detected by routine clinical cultures. The lack of a standardized system to track tissue products in healthcare facilities limits the ability of public health agencies to respond to outbreaks and investigate other adverse events associated with these products.

Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012.

Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 nasopharyngeal swab specimens collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens, with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed that all 141 tested to possess the genotype associated with macrolide susceptibility.

Legionnaires' Disease in South Africa, 2012-2014.

During June 2012-September 2014, we tested patients with severe respiratory illness for Legionella spp. infection and conducted a retrospective epidemiologic investigation. Of 1,805 patients tested, Legionella was detected in samples of 21 (1.2%); most were adults who had HIV or tuberculosis infections and were inappropriately treated for Legionella.

Investigations of Mycoplasma pneumoniae infections in the United States: trends in molecular typing and macrolide resistance from 2006 to 2013.

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 infections that were supported by the Centers for Disease Control and Prevention (CDC) between 2006 and 2013. We examined 199 M. pneumoniae-positive specimens collected during this time period in order to identify trends in antimicrobial resistance and circulating types. Overall, macrolide resistance was identified in approximately 10% of M. pneumoniae infections occurring during this time period. Typing of strains revealed cocirculation of multiple multilocus variable-number tandem-repeat analysis (MLVA) and P1 types throughout this period, including diversity in types detected within individual outbreaks. Three MLVA types (4572, 3562, and 3662) accounted for 97% of the infections during the study period. A systematic surveillance program is necessary to understand the burden of M. pneumoniae disease in the United States, facilitate case and outbreak identification, and inform appropriate therapeutic and infection control strategies.

Detection and characterization of Mycoplasma pneumoniae during an outbreak of respiratory illness at a university.

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.

Notes from the field: atypical pneumonia in three members of an extended family - South Carolina and north Carolina, july-august 2013.

On August 5, 2013, the South Carolina Department of Health and Environmental Control was notified of a case of acute respiratory failure in a previously healthy woman. A family interview revealed the patient's uncle and cousin had also been hospitalized with similar symptoms in North Carolina. The South Carolina Department of Health and Environmental Control and the North Carolina Division of Public Health collaborated to identify the cause of the respiratory illness cluster and to prevent additional illnesses.

Trypanosoma cruzi in domestic and wild mammals in the northeast region of Colombia.

Trypanosoma cruzi hosts can serve as a source of infection for animals, vectors, and humans, contributing to the establishment of Chagas disease (CD) in a given area. Traditionally, the Department of Córdoba has not been considered a transmission area for CD; however, the report of several acute cases of Chagas disease highlights the importance of studying the dynamics of disease transmission in this region. This study aimed to detect T. cruzi in domestic and wild mammals in the department of Córdoba. In 2017, a cross-sectional descriptive study was conducted in six villages in two municipalities in the department of Córdoba. Blood samples from dogs living in the zones were collected in EDTA vacutainer tubes for domestic mammals. Wild mammals were collected using Sherman and Tomahawk traps and mist nets in crops and peridomiciles. T. cruzi DNA was detected using the kinetoplast DNA (kDNA) variable region and the tandem repeat satellite region of T. cruzi as molecular targets. We sampled 168 dogs and 146 wild mammals. The detected prevalence of T. cruzi was 6.37%; the TcI lineage was found in D. marsupialis, H. anomalus, and one canine. A specimen of D. marsupialis with TcI and TcII lineages was also identified. T. cruzi DNA was detected in domestic and wild animals in the study area, indicating the circulation of the parasite in peridomestic environments. D. marsupialis may represent an important host in maintaining this region's wild and domestic cycle.

Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1.

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.

Genomic analysis of Chlamydia psittaci from a multistate zoonotic outbreak in two chicken processing plants.

Four Chlamydia psittaci isolates were recovered from clinical specimens from ill workers during a multistate outbreak at two chicken processing plants. Whole genome sequencing analyses revealed high similarity to C. psittaci genotype D. The isolates differed from each other by only two single nucleotide polymorphisms, indicating a common source.

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.

Multilocus variable-number tandem-repeat analysis of Mycoplasma pneumoniae clinical isolates from 1962 to the present: a retrospective study.

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.

Complete Genome Sequence of Mycoplasma pneumoniae Type 2 Reference Strain FH Using Single-Molecule Real-Time Sequencing Technology.

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.

Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.

We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.

Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

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.

Correction: Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

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