Calibrated Real-Time PCR for Evaluation of Parvovirus B19 Viral Load

Abstract

tion rates than those with moderate impairment (P Ͻ0.05, Mann-Whitney rank-sum test). Furthermore, in patients with severe renal impairment, there was a linear relationship between the urinary free cortisol excretion rate and CrCl (r 2 ϭ 0.83, linear regression; Fig. 1B). On the other hand, we observed no linear relationship between urinary cortisol excretion rates and random total serum cortisol or the degree of proteinuria in the studied patients (data not shown). In contrast to the strong relationship between CrCl and urinary cortisol excretion rate, there was only a weak correlation between the volume of the 24-h urine collection and urinary free cortisol (r 2 ϭ 0.16, Spearman correlation). Therefore, it is unlikely that the observed reduction in urinary free cortisol excretion was attributable to the incomplete collection of 24-h urine.Because the estimation of CrCl involves collection of urine over 24 h, we further investigated whether the reduced urinary cortisol excretion could be predicted by use of the serum creatinine concentration alone. We performed a ROC curve analysis to determine the cutoff value for serum creatinine concentrations for the prediction of CrCl of Ͻ60 mL/min and found that the cutoff values for males and females were 126 and 94 mol/L, respectively. The distributions of urinary free cortisol excretion rates and CrCl in patients with normal and increased serum creatinine concentrations are shown in Fig. 1A. The mean urinary free cortisol excretion rates in individuals with normal and increased serum creatinine concentrations were 168 and 61 nmol/day, respectively (P Ͻ0.001, Mann-Whitney rank-sum test).Our study has shown that CrCl is a major determinant for urinary cortisol excretion rate in patients with severe renal impairment. Because there is a theoretical possibility that the low urinary cortisol excretion rate may reflect the low blood cortisol concentrations in these patients, we compared the total random morning serum cortisol concentrations for patients with different degrees of renal impairment and found that serum total cortisol was significantly higher in patients with CrCl Ͻ20 mL/min (P ϭ 0.002, Kruskal-Wallis test). This finding is consistent with previous reports that the serum cortisol concentrations of patients with end stage renal failure were higher than those of healthy individuals (9 ). Therefore, it is unlikely that the observed decrease in urinary free cortisol excretion in patients with moderate to severe renal impairment was attributable to hypoadrenalism.In summary, the urinary free cortisol excretion rate is significantly reduced in patients with moderate to severe renal impairment (CrCl Ͻ60 mL/min) and consequently has diminished sensitivity in the diagnosis of Cushing syndrome in such patients. Moreover, this diminished urinary free cortisol excretion rate could be accurately predicted by the increased serum creatinine concentrations. Therefore, it would be more appropriate to use late-night serum salivary cortisol or other blood-based diagnostic tests for...

Gestational and Fetal Outcomes in B19 Maternal Infection: a Problem of Diagnosis

“…Fluorescence emissions were recorded in the FAM/Sybr channel of the instrument and analyzed by using the functions available in the RotorGene 6.0 software. B19 DNA quantitation was performed by using the algorithm described previously (11) and was expressed as B19 DNA IU/ml of sample. For the in situ hybridization assay, paraffin-embedded sections of placental biopsy specimens were dewaxed sequentially in two changes of fresh xylene, washed in absolute ethanol, and then analyzed as described previously (1).…”

Keeping Pace with Parvovirus B19 Genetic Variability: a Multiplex Genotype-Specific Quantitative PCR Assay

“…A 100-l volume of sample was processed by following the manufacturer's instructions, and nucleic acids were collected in a 100-l volume of eluate. Plasmid pEC01 (16), containing as an insert a synthetic sequence, was added at 10 9 copies/ml to the lysis buffer during the procedure, copurified with sample nucleic acids, and used as the analytical control reagent in the lab-developed qPCR assay. Conversely, in the derived protocol exploiting the QuantiFast pathogen PCR kit (Qiagen), the highly concentrated internal control DNA supplied by Qiagen was added to the lysis buffer at a ratio of 0.1 l per 1 l of sample elution.…”

“…The international NIBSC standard panel (NIBSC 99/110), comprising genotype 1, genotype 2, and genotype 3a B19V-positive plasma sam- ples, was used for assay setup and validation. Subsequently, the routine performance of the newly developed qPCR assay was evaluated by comparison to an established, validated lab-developed qPCR assay, amplifying the same target region but specific for B19V genotype 1 (16). Bioinformatic analysis.…”

“…Assay on clinical specimens. A total of 130 consecutive serum samples sent to the laboratory with a specific request of B19V PCR were analyzed with the newly developed qPCR assay, and results were compared to a reference assay (16). For each test run, a 10 3 IU/ml genotype 1 standard (NIBSC 99/802) was included as a reference sample.…”

Parvovirus B19 Infection Localized in the Intestinal Mucosa and Associated with Severe Inflammatory Bowel Disease

“…f Presence of B19 virus DNA determined and quantitated by a calibrated real-time quantitative PCR (qPCR) assay; values are expressed by using the international standard NIBSC 99/800 as the reference sample (6). The detection threshold of the assay is 1.00 ϫ 10 2 IU/ml.…”