New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections

Abstract

The human erythrovirus B19, a member of the family Parvoviridae, causes a broad and seemingly expanding spectrum of disorders (7,12,23,44). The clinical picture depends on the immune status and age of the patient (14,36,43). Parvovirus B19 shows a remarkable tropism for erythroid progenitor cells in the bone marrow, which is partly based on binding to its receptor, the blood group P antigen (6). Viral replication, and possibly also the induced immune response, interferes with physiological functions and loss of the erythroid progenitor cells, resulting in a usually subclinical reticulocytopenia (8). In patients with an underlying hematologic disorder and high blood cell turnover (e.g., hemolytic anemia), transient aplastic anemia may ensue. Immunocompromised patients, who have an increased risk of developing a persistent B19 infection, are threatened by a chronic reticulocytopenic anemia, also known as pure red cell anemia (2,8).Diagnosis of uncomplicated cases of acute B19 infection (fifth disease or arthropathy) is usually clinically based and can be accomplished by detection of specific immunoglobulin M (IgM) antibodies except in immunocompromised patients, who are prone to persistent infection and who may generate IgMspecific B19 antibodies less reliably (7,19). Likewise, specific IgG is not a reliable marker for discriminating a reconvalescent status from chronic persistent infection (35), although recent data indicate that IgG antibodies specific for nonstructural protein 1 (NS-1) of B19 are more frequently associated with persistent infection (24). Detection of DNA by hybridization or PCR has been reported to be superior in the diagnosis of prenatal B19 infections and in children with oncologic or hematologic disorders (5,11,34). Several qualitative and quantitative PCR methods targeting different regions of the parvovirus genome have been published (1,4,9,10,13,15,17,22,25,40). These PCR approaches are based on conventional block thermocycling and combined single-round PCRs with subsequent oligohybridization or use a nested format.The recently developed LightCycler (LC) DNA amplification technology (Roche Diagnostics, Mannheim, Germany) combines rapid glass capillary thermal cycling with real-time microvolume fluorescence monitoring (47). Detection of amplicons is achieved during the run in real time. Melting point analysis of the amplicons at the end of the run is used as a specificity control when the fluorochrome SYBR green is used for detection of double-stranded DNA. Alternatively, specificity can be tested during the run by using two target-specific hybridization probes which utilize fluorescence resonance energy transfer (FRET) to generate a measurable signal. In the latter case, two oligonucleotide probes (HybProbe) bind to immediately adjacent regions of the respective amplicon. The upstream probe (referred to as the probe) is labeled at its 3Ј end with fluorescein, while the 5Ј end of the downstream probe (referred to as the anchor) is labeled with either of the fluorochromes LC-Red 640 and LC-...

Identification and Characterization of Persistent Human Erythrovirus Infection in Blood Donor Samples

“…This discrepancy was clearly unrelated to sample origin but rather reflected differences in the sensitivity of the assays used in the different studies. The nucleic acid testing (NAT) assays developed in the present study had a higher sensitivity (25 to 50 IU/ml) and a broader linear range (50 to 10 10 IU/ml) than in-house or commercial assays previously described (detection limit, Ն100 IU/ml; linear range over 7 log units) (2,13,22,33). Testing minipools of 10 instead of 50 to 1,200 plasma samples also affected detection.…”

Detection and Differentiation of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Isolates by Real-Time PCR

“…The temperature at which the hybridization probes dissociated from their target sites was determined by melting curve analysis. This allowed for differentiation between species based on differences in the avidity of the hybridization probes for the complementary sequences in the amplified (24) Clinical isolates GC, BR, AP, LiPA, PS M. avium (17) Clinical isolates GC, BR, AP, PS M. intracellulare (8) Clinical isolates GC, BR, AP, PS M. marinum (22) Clinical isolates GC, BR, PS M. gordonae (17) Clinical isolates GC, BR, AP M. fortuitum (19) Clinical DNA. The melting curve for each specimen was analyzed manually to determine the melting temperature (T m ).…”

Evaluation of the Roche LightCycler parvovirus B19 quantification kit for the diagnosis of parvovirus B19 infections

“…Similarly, in patient 2 (Table 3), the LC results showed how viral clearance could be monitored quantitatively. LC assay for B19 DNA has also been used to monitor the outcome where attenuating immunosuppression of the underlying disease, allowing the patient to mount an immune response, was used in the management of relapsing parvovirus B19 infection in an immunocompromised patient (Harder et al, 2001).…”

“…However, it was not until the development of real-time PCR techniques that convenient and rapid quantification of B19 DNA became feasible. Glass capillary thermal cycling (LightCyler, LC) methods with detection by either DNA-binding dye (Manaresi et al, 2002) or hybridisation probes (Harder et al, 2001) have been described as well as hydrolysis probe-based assays (TaqMan, Aberham et al, 2001). A commercial LC kit for parvovirus B19 (Roche Diagnostics) has recently become available that, as an additional feature, includes an internal control to detect any interference from inhibitory substances in the specimen.…”

“…Quantitative assays are therefore required to identify and remove plasma donations with high B19 viral load to prepare plasma pools that are safe with respect to transmission of parvovirus B19. In the diagnostic setting, quantitative measurement of B19 DNA is important for clinical management because it provides a means of monitoring the response to therapy of severe B19 infections in immunocompromised patients (Azzi et al, 1996;Harder et al, 2001). It also has a role in confirming congenital parvovirus B19 infection (Knöll et al, 2002).…”