Using PCR-Based Detection and Genotyping to Trace Streptococcus salivarius Meningitis Outbreak Strain to Oral Flora of Radiology Physician Assistant

We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis

Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract

We performed culture-based and PCR-based tests for pneumococcal identification and serotyping from carriage specimens collected in rural and urban Kenya. Nasopharyngeal specimens from 237 healthy children 98.7%) with the reference cmPCR amplicon for the st, while cmPCR amplicons from lytA-negative specimens were generally more divergent. Separate testing of 56 A-OPs and 56 A-NPs revealed that ∼94% of the positive cmPCR results from A-NP/OPs were from OP microbiota. In contrast, A-NPs yielded >2-fold more pneumococcal isolates than A-OPs. Verified and suspected non-pneumococcal cmPCR serotypes/serogroups appeared to be relatively rare in C-NPs and A-NPs compared to A-OPs. Our findings indicate that non-pneumococcal species can confound serotype-specific PCR and other sequence-based assays due to evolutionarily conserved genes most likely involved in biosynthesis of surface polysaccharide structures

Multilocus allele and 16S rRNA gene sequence designation depicting comparison of <i>S. salivarius</i> strain from meningitis patient to oral carriage isolates recovered from radiology physician assistant and technician.^a

a<p>The 8 housekeeping gene targets <i>glcK</i>, <i>ddlA</i>, <i>pepO</i>, <i>ilvC</i>, <i>thrS</i>, <i>pyrE</i>, <i>dnaE</i>, and <i>sodA</i> refer to the <i>S. salivarius</i>group MLST scheme described in reference 5 and the alleles correspond to the same ones listed in reference 5 except where in bold and italicized. The 7 remaining housekeeping gene targets (<i>rpoB</i>, <i>sodA2</i>, <i>pyk</i>, <i>ppaC</i>, <i>tuf</i>, <i>pfl</i>, <i>map</i>) refer to the viridans streptococcal MLST/speciating scheme available at <a href="http://viridans.emlsa.net/" target="_blank">http://viridans.emlsa.net/</a> (5). All bold/italicized designations correspond to sequences not found in the GenBank that were found in these isolates. GenBank accessions for the sequences corresponding to all of the underlined numbers are supplied in the Methods. The 8 following <i>S. salivarius</i> sequences have been previously documented by other investigators and are listed along with their GenBank accession numbers: <i>rpoB1</i>/GU556187, <i>sodA2-1</i>/AB200166, <i>ppaC1</i>/GU556189, <i>pyk2</i>/CP002888, <i>tuf1</i>/GU556190, <i>tuf2</i>/CP002888, 16S <i>rrnA2</i>/CP002888, and 16S <i>rrnA1</i>/GU175444.</p>b<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032169#s3" target="_blank">Results</a> shared between two isolates from case CSF.</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032169#s3" target="_blank">Results</a> shared between three carriage isolates (dorsal tongue and saliva).</p>d<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032169#s3" target="_blank">Results</a> shared between two carriage isolates (dorsal tongue and saliva).</p

Multisystem Inflammatory Syndrome in U.S. Children and Adolescents.

BACKGROUND: Understanding the epidemiology and clinical course of multisystem inflammatory syndrome in children (MIS-C) and its temporal association with coronavirus disease 2019 (Covid-19) is important, given the clinical and public health implications of the syndrome. METHODS: We conducted targeted surveillance for MIS-C from March 15 to May 20, 2020, in pediatric health centers across the United States. The case definition included six criteria: serious illness leading to hospitalization, an age of less than 21 years, fever that lasted for at least 24 hours, laboratory evidence of inflammation, multisystem organ involvement, and evidence of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on reverse-transcriptase polymerase chain reaction (RT-PCR), antibody testing, or exposure to persons with Covid-19 in the past month. Clinicians abstracted the data onto standardized forms. RESULTS: We report on 186 patients with MIS-C in 26 states. The median age was 8.3 years, 115 patients (62%) were male, 135 (73%) had previously been healthy, 131 (70%) were positive for SARS-CoV-2 by RT-PCR or antibody testing, and 164 (88%) were hospitalized after April 16, 2020. Organ-system involvement included the gastrointestinal system in 171 patients (92%), cardiovascular in 149 (80%), hematologic in 142 (76%), mucocutaneous in 137 (74%), and respiratory in 131 (70%). The median duration of hospitalization was 7 days (interquartile range, 4 to 10); 148 patients (80%) received intensive care, 37 (20%) received mechanical ventilation, 90 (48%) received vasoactive support, and 4 (2%) died. Coronary-artery aneurysms (z scores ≥2.5) were documented in 15 patients (8%), and Kawasaki\u27s disease-like features were documented in 74 (40%). Most patients (171 [92%]) had elevations in at least four biomarkers indicating inflammation. The use of immunomodulating therapies was common: intravenous immune globulin was used in 144 (77%), glucocorticoids in 91 (49%), and interleukin-6 or 1RA inhibitors in 38 (20%). CONCLUSIONS: Multisystem inflammatory syndrome in children associated with SARS-CoV-2 led to serious and life-threatening illness in previously healthy children and adolescents. (Funded by the Centers for Disease Control and Prevention.)