c Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log 10 versus 4 log 10 copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R 2 > 0.98 in each of 6 regression models) and clinical samples (R 2 ‫؍‬ 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

BACKGROUND: Viral load testing for cytomegalovirus (CMV) is an important diagnostic tool for the management of transplant recipients and immunocompromised individuals; however, inconsistency among laboratories in quantitative measurements of viral load limits interinstitutional comparisons. These inconsistencies stem from the lack of assays cleared by the US Food and Drug Administration, the absence of international standards, the wide variety of CMV-extraction and -detection methods, and differences in materials used for calibration. A critical component of standardization is the use of calibrators that are traceable and commutable.

Accurate and rapid diagnosis is needed for timely intervention and clinical management of acute respiratory infections. This study evaluated performance characteristics of the Panther Fusion assay for the detection of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3 (Para 1 to 3), human metapneumovirus (hMPV), rhinovirus (RV), and adenovirus (Adeno) targets in comparison to those of the eSensor and Lyra assays using 395 nasopharyngeal (NP) and 104 lower respiratory tract (LRT) specimens. Based on the consensus positive result established (positive result in 2 of the 3 assays), the NP specimens for the Fusion and eSensor assays had 100% positive percent agreement (PPA) for all the analytes and the Lyra assays had 100% PPA for Flu A and Adeno analytes. A 100% negative percent agreement (NPA) was observed for all the Lyra analytes, whereas those for the Fusion targets ranged from 98.4 to 100% and those for the eSensor ranged from 99.4 to 100% for all the analytes except RV. For the LRT specimens, Fusion had 100% PPA and 100% NPA for all the targets except hMPV. There was a 100% PPA for eSensor analytes; the NPA ranged from 98 to 100%, except for RV. For the Lyra assays, the PPA ranged between 50 and 100%, while the NPA was 100% for all the targets except Adeno. The Fusion assay performed similarly to the eSensor assay for majority of the targets tested and provides laboratories with a fully automated random-access system to test for a broad array of viral respiratory pathogens.

Trichomonas vaginalis is an important pathogen in both men and women. Culture is considered the diagnostic gold standard, although studies have shown that PCR is more sensitive than either culture or wet mount for the diagnosis of T. vaginalis infections. We sought to identify a simple method for stabilizing T. vaginalis DNA in urine samples that could be easily applied to molecular testing. The stability of T. vaginalis DNA in 40 urine samples was assessed by storage for various times at either 4°C or room temperature with or without the Becton Dickinson urine preservative transport (UPT) kit. Overall, there was better stability of T. vaginalis DNA when specimens were stored at 4°C than when they were stored at 20 to 22°C and when the UPT system was used. T. vaginalis DNA was stable in specimens stored without using the UPT at 4°C for about 3 days and at room temperature for only 1 day. For specimens placed in the UPT within 24 h (times of 1, 6, and 24 h) of collection, the DNA was stable for up to 30 days when stored at 4°C. For specimens stored at room temperature, the urine should be added to the UPT ideally within 1 hour of collection, and in this case the DNA remained stable for up to 30 days. When storing specimens at room temperature, a delay of 24 h prior to adding to UPT led to an unacceptably high loss of assay sensitivity.Trichomonas vaginalis is a common sexually transmitted disease, with an estimated 7.4 million new cases annually in the United States and as many as 180 million cases worldwide (13). T. vaginalis can cause symptoms in both men and women and can cause severe complications in pregnant women. Symptoms in women include vaginal discharge and vulval irritation; if untreated, more-severe complications may occur, including endometritis, cervical erosion, and infertility (3, 9). T. vaginalis infections in pregnant women can lead to premature rupture of membranes, low-birth-weight infants, and preterm deliveries (2). In men, infection with T. vaginalis may cause nongonococcal urethritis, with untreated infections causing chronic prostatitis, epididymitis, infertility, or urethral strictures (3, 9). Moreover, T. vaginalis infection can lead to an increased risk of acquiring human immunodeficiency virus type 1 infection due to the local inflammation caused by T. vaginalis infections in the genital tract (5,7,8,11).There are numerous reports supporting the role of PCR in the diagnosis of T. vaginalis infections from genital specimens; these reports have shown that the detection of T. vaginalis DNA by PCR is significantly more sensitive than both culture and wet mount examination (1,4,6,12,14,15). We have previously reported that vaginal swabs collected with the BDProbeTec dry swab system were a reliable specimen for PCR testing and allowed the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and T. vaginalis from a single specimen (1). For clinical studies using urine specimens, our procedure for stabilizing T. vaginalis was cumbersome and involved adding an aliquot of urine to Fuji me...

Twelve laboratories evaluated candidate material for an Aspergillus DNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 10 10 units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid.