The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. That non-classical monocytes may be a source of inflammation in PASC warrants further study.

Background Repeat COVID-19 molecular testing can lead to positive test results after negative tests and to multiple positive test results over time. The association between positive tests and infectious virus is important to quantify. Methods A two months cohort of retrospective data and consecutively collected specimens from COVID-19 patients or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of SARS-CoV-2 in cell culture. Whole genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital PCR (ddPCR) was used to assess the rate of false negative COVID-19 diagnostic tests. Results In two months, 29,686 specimens were tested and 2,194 patients received repeated testing. Virus recovery in cell culture was noted in specimens with SARS-CoV-2 target genes’ Ct value average of 18.8 ± 3.4. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 20 days after the first positive result but mostly in individuals symptomatic at time of sample collection. Whole genome sequencing provided evidence the same virus was carried over time. Positive tests following negative tests had Ct values higher than 29.5 and were not associated with virus culture. ddPCR was positive in 5.6% of negative specimens collected from COVID-19 confirmed or clinically suspected patients. Conclusions Low Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Enumerating Aspergillus fumigatus CFU can be challenging since CFU determination by plate count can be difficult. CFU determination by quantitative real-time PCR (qPCR), however, is becoming increasingly common and usually relies on detecting one of the subunits of the multicopy rRNA genes. This study was undertaken to determine if ribosomal DNA (rDNA) copy number was constant or variable among different A. fumigatus isolates. FKS1 was used as a single-copy control gene and was validated against single-copy (pyrG and ARG4) and multicopy (arsC) controls. The copy numbers of the 18S rDNA subunit were then determined for a variety of isolates and were found to vary with the strain, from 38 to 91 copies per genome. Investigation of the stability of the 18S rDNA copy number after exposure to a number of different environmental and growth conditions revealed that the copy number was stable, varying less than one copy across all conditions, including in isolates recovered from an animal model. These results suggest that while the ribosomal genes are excellent targets for enumeration by qPCR, the copy number should be determined prior to using them as targets for quantitative analysis.

Subsequent to the publication of our article, it was recognized that some details of our PCR and sequencing procedures were not thoroughly described. Page 4016, column 1: Lines 17-30 should read as follows. "Amplicons to be sequenced (high-fidelity PCRs) were prepared using Triple Master Taq polymerase (Eppendorf, Westbury, NY) according to the manufacturer's instructions. The high-fidelity PCRs consisted of a final concentration of 2.5 units of Taq polymerase, 5 l of the template DNA, 200 nM of each primer, 200 M dNTP, 1ϫ buffer with magnesium, and distilled water to achieve a final volume of 50 l. Screening PCRs (nonsequenced) were prepared with Taq polymerase from Invitrogen (Carlsbad, CA) using component concentrations recommended by the manufacturer for the basic PCR protocol. Reactions were run in an MJ mini thermocycler (Bio-Rad Laboratories, Hercules, CA), using a basic cycling program of 94°C for 2 min (first cycle only), 94°C for 15 s, the primer-specific annealing temperature (see references from Table 1) for 30 s, 72°C for 30 s (30 cycles), and 72°C for 3 min. The primers are shown in Table 1 and were synthesized at the Advanced Nucleic Acids Core Facility at the University of Texas Health Science Center at San Antonio. The primers for CTX-M group 3 were redesigned (CTX-M8.WSAgroupIII.F and CTX-M8.WSAgroupIII.R; annealing temperature, 65°C) using sequences from GenBank (accession numbers X92506, AF189721, X92507, and AF252622)." Page 4016, column 2: Lines 4-13 should read as follows. "The predicted PCR product was a 540-bp intragenic fragment of bla OXA-1. PCRs were performed in a DNA thermal cycler (Eppendorf) and prepared as described above using Triple Master Taq polymerase (Eppendorf). Thirty-five cycles were performed for each reaction using the following temperature profile: 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min. All PCR products from the isolates were sequenced. The sequence of the bla OXA-30 gene was deposited under Genbank accession number AF255921.

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