High-level JCPyV viruria after kidney transplantation-Clinical and histopathological findings.

BACKGROUND: The significance of JC polyomavirus (JCPyV) after kidney transplantation ranges from irrelevant to full-blown nephropathy or PML. OBJECTIVES: To investigate the clinical significance of high-level JCPyV viruria and JCPyV primary infections after kidney transplantation. STUDY DESIGN: JCPyV viruria was detected in routine screening by quantitative real-time PCR in 40/238 kidney transplant recipients and was high-level (>107 copies/ml) in 17 patients. A protocol biopsy at the time of JCPyV viruria was available from 10 patients. RESULTS: Peak urine viral loads were 1.0×107-2.5×109 copies/ml in the 17 high-level viruria patients. 6/15 (40%) patients with high-level JCPyV viruria with pretransplant sera available were JCPyV IgG negative suggesting that JCPyV viruria resulted from the donor graft in most cases. No acute graft dysfunction was associated with JCPyV viruria. No positive SV40 staining was detected in protocol biopsies, and no specific histopathology was associated with high-level viruria; JCPyV nephropathy was not found. No differences were seen in histopathology or graft function at 3 years in patients with high-level viruria compared to non-JCPyV viruric patients transplanted during the same time period, and outcome was similar in patients with presumably primary and reactivated JCPyV. The mean estimated GFR at last follow-up was 44ml/min (range 12-60ml/min). One graft with high-level viruria was lost 9 years posttransplant due to recurrent IgA nephropathy CONCLUSIONS: High-level JCPyV viruria seems to be associated with primary JCPyV infection reflecting the average seroprevalence of 60%, but is not stringently associated with inferior graft function or survival, or histopathological changes.

Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human ß-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.

Expression of BKV and JCV encoded microRNA in human cerebrospinal fluid, plasma and urine.

BACKGROUND: BK and JC polyomaviruses encode microRNAs which may facilitate the establishment of persistent infection. MicroRNAs contribute to disease pathogenesis, and may provide useful tools in laboratory diagnostics and patient management. OBJECTIVES: In this pilot work we studied whether viral and cellular microRNAs can be extracted and detected from body fluids to provide added value in a diagnostic laboratory. STUDY DESIGN: Altogether 120 human plasma, urine, and cerebrospinal fluid samples from individuals diagnosed with, or suspected of, a severe polyomavirus associated disease, were included in the study. The samples were spiked with unrelated synthetic microRNA to control for sample quality and inhibition. BKV specific bkv-miR-B1-5p, JCV specific jcv-miR-J1-5p, and bkv-miR-B1-3p/jcv-miR-J1-3p, sharing identical sequences between the two viruses, were amplified from human samples using specific TaqMan assays. Expression of 84 circulating human microRNAs was studied in four selected plasma samples in microarray. RESULTS: jcv-miR-J1-5p and bkv-miR-B1-3p/jcv-miR-J1-3p were frequently amplified from human plasma, urine, and cerebrospinal fluid samples. bkv-miR-B1-5p was amplified from one-third of the samples, which often contained high viral DNA loads. A microarray screen of human microRNAs in plasma samples suggested regulation of several human microRNA expression in BKV positive vs negative samples. CONCLUSIONS: Viral and cellular microRNAs can be processed and detected from human body fluids. They may prove useful in the diagnosis and management of severe polyomavirus associated diseases, calling for further clinical evaluation.