Matrix Matters: Assessment of Commutability among BK Virus Assays and Standards.

Quantitative testing of BK virus (BKPyV) nucleic acid has become the standard of care in transplant patients. While the relationship between interassay harmonization and commutability has been well characterized for other transplant-related viruses, it has been less well studied for BKPyV, particularly regarding differences in commutability between matrices. Here, interassay agreement was evaluated among six real-time nucleic acid amplification tests (NAATs) and one digital PCR (dPCR) BKPyV assay. Differences in the commutability of three quantitative standards was examined across all assays using a variety of statistical approaches. Panels, including 40 samples each of plasma and urine samples previously positive for BKPyV, together with one previously negative plasma sample and four previously negative urine samples, were tested using all assays, with each real-time NAAT utilizing its usual quantitative calibrators. Serial dilutions of WHO, National Institute for Standards and Technology (NIST), and commercially produced (Exact/Bio-Rad) reference materials were also run by each assay as unknowns. The agreement of the clinical sample values was assessed as a group and in a pairwise manner. The commutability was estimated using both relativistic and quantitative means. The quantitative agreement across assays in the urine samples was within a single log10 unit across all assays, while the results from the plasma samples varied by 2 to 3 log10 IU/mL. The commutability showed a similar disparity between the matrices. Recalibration using international standards diminished the resulting discrepancies in some but not all cases. Differences in the sample matrix can affect the commutability and interassay agreement of quantitative BKPyV assays. Differences in commutability between matrices may largely be due to factors other than those such as amplicon size, previously described as important in the case of cytomegalovirus. Continued efforts to standardize viral load measurements must address multiple sources of variability and account for differences in assay systems, quantitative standards, and sample matrices.

Immune signatures underlying post-acute COVID-19 lung sequelae.

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)­specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

Impact of Fragmentation on Commutability of Epstein-Barr Virus and Cytomegalovirus Quantitative Standards.

Despite the adaptation of international standards, quantitative viral load testing of transplant-associated viruses continues to be limited by interlaboratory disagreement. Studies have suggested that this disagreement and the poor commutability of standards may, in some cases, be linked to amplicon size and the fragmentation of circulating viral DNA. We evaluated target fragmentation as a cause of noncommutability and pretest fragmentation of quantitative standards as a potential means of increasing commutability and interassay agreement. Forty-two cytomegalovirus (CMV)-positive and 41 Epstein-Barr virus (EBV)-positive plasma samples, together with two different quantitative standards for each virus, were tested as unknowns using 10 different quantitative PCR assays at 5 different laboratories. Standards were tested both intact and after intentional fragmentation by ultrasonication. Quantitative agreement between methods was assessed, together with commutability, using multiple statistical approaches. Most assays yielded results within 0.5 log10 IU/ml of the mean for CMV, while for EBV a greater variability of up to 1.5 log10 IU/ml of the mean was shown. Commutability showed marked improvement following fragmentation of both CMV standards but not after fragmentation of the EBV standards. These findings confirm the impact of amplicon size and target fragmentation on commutability for CMV and suggest that for some (but not all) viruses, interlaboratory harmonization can be improved through the use of fragmented quantitative standards.

Hospital-acquired listeriosis linked to a persistently contaminated milkshake machine.

One case of hospital-acquired listeriosis was linked to milkshakes produced in a commercial-grade shake freezer machine. This machine was found to be contaminated with a strain of Listeria monocytogenes epidemiologically and molecularly linked to a contaminated pasteurized, dairy-based ice cream product at the same hospital a year earlier, despite repeated cleaning and sanitizing. Healthcare facilities should be aware of the potential for prolonged Listeria contamination of food service equipment. In addition, healthcare providers should consider counselling persons who have an increased risk for Listeria infections regarding foods that have caused Listeria infections. The prevalence of persistent Listeria contamination of commercial-grade milkshake machines in healthcare facilities and the risk associated with serving dairy-based ice cream products to hospitalized patients at increased risk for invasive L. monocytogenes infections should be further evaluated.

Multicenter evaluation of a commercial cytomegalovirus quantitative standard: effects of commutability on interlaboratory concordance.

Commutability of quantitative reference materials has proven important for reliable and accurate results in clinical chemistry. As international reference standards and commercially produced calibration material have become available to address the variability of viral load assays, the degree to which such materials are commutable and the effect of commutability on assay concordance have been questioned. To investigate this, 60 archived clinical plasma samples, which previously tested positive for cytomegalovirus (CMV), were retested by five different laboratories, each using a different quantitative CMV PCR assay. Results from each laboratory were calibrated both with lab-specific quantitative CMV standards ("lab standards") and with common, commercially available standards ("CMV panel"). Pairwise analyses among laboratories were performed using mean results from each clinical sample, calibrated first with lab standards and then with the CMV panel. Commutability of the CMV panel was determined based on difference plots for each laboratory pair showing plotted values of standards that were within the 95% prediction intervals for the clinical specimens. Commutability was demonstrated for 6 of 10 laboratory pairs using the CMV panel. In half of these pairs, use of the CMV panel improved quantitative agreement compared to use of lab standards. Two of four laboratory pairs for which the CMV panel was noncommutable showed reduced quantitative agreement when that panel was used as a common calibrator. Commutability of calibration material varies across different quantitative PCR methods. Use of a common, commutable quantitative standard can improve agreement across different assays; use of a noncommutable calibrator can reduce agreement among laboratories.

Key gravity-sensitive signaling pathways drive T cell activation.

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity.

Early immune response and regulation of IL-2 receptor subunits.

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

CD14-mediated alterations in transcription and splicing of endogenous retroviruses after injury.

Increase in systemic levels of lipopolysaccharide (LPS) contributes to the pathogenesis of distant organ injury after burn. Stress signals elicited from burn influence transcriptional activities of mouse endogenous retroviruses (MuERVs) in various distant organs. The involvement of LPS pathways in the burn-mediated regulation of MuERVs in the spleen was investigated in this study. Spleen harbors substantial numbers of tissue macrophages, a key responder to LPS stimulation. Spleen tissues collected from CD14 (LPS receptor) knockout (KO) and wild type (WT) mice after burn were subjected to RT-PCR analysis of MuERV expression. There was a substantial induction of 2 bands and a marked downregulation of a band in CD14 KO mice compared to WT mice after burn. Sequence analysis of these CD14- and burn-dependent bands identified 3 new alternatively spliced and 2 defective env transcripts of MuERVs as well as novel splicing signals. Chromosomal loci of putative MuERVs sharing the unique U3 sequences of these transcripts were mapped by surveying the entire genome of C57BL/6J mice. In addition, coding potentials, transcriptional regulatory elements, and adjacent cellular genes of these putative MuERVs were analyzed. The results from these studies suggest that injury-triggered LPS/CD14 signaling events play roles in the transcriptional regulation of certain MuERVs carrying unique U3 promoter sequences.