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Rationale & Objective: The nasal passages harbor both commensal and pathogenic bacteria. In this study, we sought to characterize the anterior nasal microbiota in PD patients using 16S rRNA gene sequencing. Study Design: Cross-sectional. Setting & Participants: We recruited 32 PD patients, 37 kidney transplant (KTx) recipients, 22 living donor/healthy control (HC) participants and collected anterior nasal swabs at a single point in time. Predictors: We performed 16S rRNA gene sequencing of the V4-V5 hypervariable region to determine the nasal microbiota. Outcomes: Nasal microbiota profiles were determined at the genus level as well as the amplicon sequencing variant level. Analytical Approach: We compared nasal abundance of common genera among the 3 groups using Wilcoxon rank sum testing with Benjamini-Hochberg adjustment. DESeq2 was also utilized to compare the groups at the ASV levels. Results: In the entire cohort, the most abundant genera in the nasal microbiota included: Staphylococcus, Corynebacterium, Streptococcus , and Anaerococcus . Correlational analyses revealed a significant inverse relationship between the nasal abundance of Staphylococcus and that of Corynebacterium . PD patients have a higher nasal abundance of Streptococcus than KTx recipients and HC participants. PD patients have a more diverse representation of Staphylococcus and Streptococcus than KTx recipients and HC participants. PD patients who concurrently have or who developed future Staphylococcus peritonitis had a numerically higher nasal abundance of Staphylococcus than PD patients who did not develop Staphylococcus peritonitis. Limitations: 16S RNA gene sequencing provides taxonomic information to the genus level. Conclusions: We find a distinct nasal microbiota signature in PD patients compared to KTx recipients and HC participants. Given the potential relationship between the nasal pathogenic bacteria and infectious complications, further studies are needed to define the nasal microbiota associated with these infectious complications and to conduct studies on the manipulation of the nasal microbiota to prevent such complications.
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BACKGROUND: BK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay. METHODS: We determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays. The diagnostic accuracy of the Bak standard curve in the RT-qPCR assay for the noninvasive diagnosis of BKVN in human kidney allograft recipients was investigated by quantification of BKV VP1 mRNA copy number in 192 urine samples matched to 192 kidney allograft biopsies from 155 unique kidney allograft recipients. Intraclass correlation coefficients (ICC) were calculated for the threshold cycles (Ct) and BKV VP1 mRNA copy number observed in the RT-qPCR assay with the Bak standard curve or the BKV standard curve. RESULTS: Performance characteristics of the Bak amplicon-based RT-qPCR assay were exceptional with a slope of -3.291, Y-intercept of 38.60, R2 value of 1.00, efficiency of 101% and error of 0.014. Amplification was specific for the Bak amplicon. Intra assay standard deviation (SD) was 0.08 or less and inter assay SD was 0.11 or less for 31 cycles or less of amplification of the Bak amplicon. Receiver operating characteristic (ROC) curve analysis of BKV VP1 mRNA copy number in 192 biopsy matched urines yielded an area under the ROC of 0.982 (95% CI, 0.964 to 0.999, P < 0.0001) for discriminating patients with BKVN biopsies from patients without BKVN biopsies. The striking identity in the measurement of BKV VP1 mRNA copy numbers in the Bak amplicon-based RT-qPCR assay and in the BKV amplicon-based RT-qPCR assay was shown by an ICC of 1.00 when the Cts were compared, and an ICC of 0.99 when the log10 BKV VP1 mRNA copy numbers were compared. CONCLUSIONS: Bak standard curve for absolute quantification of BKV VP1 mRNA copy number in the RT-qPCR assay demonstrated high efficiency, short and long-term precision and analytical specificity. BKVN was diagnosed with high accuracy. Our new findings, viewed in the light of our earlier demonstration that absolute quantification of a panel of mRNAs encoding immunoregulatory proteins is feasible with the Bak amplicon-based RT-qPCR assays, suggest that the Bak standard curve could serve as a universal calibrator for absolute quantification of transcripts in RT-qPCR assays and help reduce the workload, costs and eliminate contamination of genes of interest by repeated amplification of gene specific standard curves.
