Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.

Polyomavirus infection and related nephropathy is being increasingly recognized as an important cause of allograft dysfunction in adult renal transplant recipients. We prospectively monitored pediatric renal transplant recipients for the presence of BK and JC polyomavirus in urine and blood using a quantitative PCR assay to evaluate the prevalence and clinical relevance of polyomavirus infection in the pediatric renal transplant population. Of 46 pediatric renal recipients who were evaluated, nine (20%) demonstrated isolated BKV viruria, while five (11%) had concomitant BKV viremia and viruria. JCV viruria was found in eight (17%) patients. BKV viremia was associated with a significantly higher urinary BKV viral load: median urinary viral load 1.9 x 10(9) copies/mL (range 6.7 x 10(2)-1.8 x 10(11)) for the group with concomitant viremia and viruria vs. 1.8 x 10(3) copies/mL (range 2.5 x 10(2)-4.5 x 10(6)) for the group with isolated viruria (p < 0.0001). In children that were followed prospectively since their transplantation, the BKV urinary viral load increased markedly before viremia became detectable a few weeks later. None of the patients with JCV viruria or isolated BKV viruria had renal dysfunction. Among the five patients with BKV in both urine and blood, two developed biopsy-proven BKV nephropathy associated with deterioration of the renal function. Management of the BKV nephropathy consisted of reduction of immunosuppression alone or in combination with antiviral treatment with cidofovir. This study shows that polyomavirus infection and related interstitial nephritis is a relevant clinical issue in the pediatric renal transplant population. Monitoring the polyomaviral load in the urine and the blood of the patients using a quantitative PCR technique is a useful tool in the diagnosis and subsequent management of this infection. Even before viremia is present, an important rise in the urinary viral load should draw the attention of the transplant clinician and raise the issue of adapting the immunosuppression.

Measuring serum beta-d-glucan (BDG) is a useful tool for supporting a quantitative PCR (qPCR)-based diagnosis of suspected Pneumocystis pneumonia (PCP) with bronchoalveolar lavage (BAL) fluid. Since the 2000s, the Fungitell assay was the only BDG assay which was FDA cleared and Conformité Européenne (CE) marked. However, the Wako β-glucan test was also recently CE marked and commercialized. We analyzed archived sera from 116 PCP cases (who were considered to have PCP based on compatible clinical and radiological findings plus a BAL fluid qPCR threshold cycle value of ≤28) and 114 controls (those with a BAL fluid qPCR threshold cycle value of >45 and no invasive fungal infection) using the Fungitell and Wako assays in parallel and assessed their diagnostic performance using the manufacturer’s proposed cutoffs of 80 pg/ml and 11 pg/ml, respectively. We found the Wako assay to be more specific (0.98 versus 0.87, P < 0.001) and the Fungitell assay to be more sensitive (0.78 versus 0.85, P = 0.039) at the proposed cutoffs. Overall performance, as determined by the area under the receiver operating characteristic curve, was similar for both assays. We determined a new Wako assay cutoff (3.616 pg/ml) to match the sensitivity of the Fungitell assay (0.88 at a cutoff of ≥60 pg/ml). Using this newly proposed cutoff, the specificity of the Wako assay was significantly better than that of the Fungitell assay (0.89 versus 0.82, P = 0.011). In conclusion, the Wako assay performed excellently compared to the Fungitell assay for the diagnosis of presumed PCP based on qPCR. In addition, contrary to the Fungitell assay, the Wako assay allows for single-sample testing with lower inter- and intrarun variability. Finally, we propose an optimized cutoff for the Wako assay to reliably exclude PCP.

The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.Human cytomegalovirus (CMV) is the leading cause of congenital viral infection in developed countries, with the reported incidence varying between 0.2 and 2.2% of all live births (15,35). The transmission rate following primary infection of the mother is about 40%. Only 10 to 15% of the CMV-infected children are symptomatic at birth, and the symptoms range from mild to life-threatening disease. The remaining 85 to 90% of the children are asymptomatic at birth, but 10% of them will develop complications later on, mainly neurodevelopmental defects and sensorineural hearing loss. Among pregnant women with recurrent infection, the rate of transmission to the infant is about 1%. Despite a preexisting immunity in the mother, epidemiological data suggest that the frequency and the severity of symptoms might be in the same range as those for a primary CMV infection (11,12).The issue of whether pregnant women should be screened for CMV during pregnancy has been debated for many years, but no consensus has been agreed upon (6). None of the current international guidelines recommend routine serologic screening of pregnant women (1,7,16,23,26). Indeed, there is no prognostic marker in the mother to predict whether the virus will be transmitted to the fetus (32). To obtain more information, invasive prenatal diagnostic techniques, such as amniocentesis or cordocentesis, have been used. Moreover, CMV infection of the fetus can lead to a great variety of clinical and biological conditions, but there is no reliable marker that can be used to predict which infected fetuses will have serious sequelae (32, 33). Finally, no vaccine or prophylactic therapy is available at present (24, 32). Nigro et al. examined whether CMV-specific hyperimmune globulin therapy could be useful for the prevention and treatment of congenital CMV infection, yet the results of the study did not allow any conclusions to be drawn (28). Despite the drawbacks of the diagnosis and treatment of a congenital CMV infection, gynecologists do screen their patients for CMV (18). Supporters of routine prenatal screening argue that the use of precautionary hygienic measures can be suggested to CMV-seronegative pregnant women. Otherwise, the k...

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples ( hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTAblood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ؎ 0.06 for HCV and 0.97 ؎ 0.03 for HBV, indicating a linear extraction from 100 to 1.0 ؋ 10 5 HCV IU/ml and from 100 to 1.0 ؋ 10 6 HBV IU/ml. Intra-and interrun variability was below 0.23 log 10 IU/ml for 2.98 to 5.28 log 10 HCV IU/ml and 2.70 to 5.20 log 10 HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log 10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log 10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log 10 copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log 10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.Real-time thermocyclers have greatly decreased the amount of hands-on time in nucleic acid (NA)-based diagnostics for pathogens. The use of commercially available universal master mixes that contain all reagents except the pathogen-specific primer-probe combination has further decreased the number of pipetting steps and thus reduced labor and the possibility of errors.NA extraction has now become the most critical and laborintensive step in NA-based diagnostic assays. The overall sensitivity of the assay is determined by the NA yield, its purity, and the amount of sample equivalents that can be transferred into the amplification reaction. Conventional manual sample preparation methods are labor intensive and susceptible to contamination, handling variations, or errors (3, 9, 12).Since both the pathogen range and the number of different sample types are expanding and since multiplex downstream testing is becoming standard practice, there is a need for a generic extraction method (2, 5, 10). Ideally, the NA extraction procedure should yield pure NA from different pathogens and from a broad range of sample types.Recently, Abbott introduced m1000, an automated generic RNA and DNA extraction system using magnetic microparticle processing (17, 18). The m1000 system provides the necessary features for full automation of NA extraction of up to...

No abstract

The level of HIV-1 RNA in plasma has become one of the most important markers in the follow-up of HIV-infected patients. Three techniques are commercially available: both the Amplicor HIV Monitor and the NASBA HIV-1 RNA QT are target amplification methods, whereas the Quantiplex HIV RNA assay is a branched DNA signal amplification technique. Detection in both target amplification techniques is based on a single primer pair and a single probe in the gag region, whereas multiple probes capture the pol region of the viral RNA in the branched DNA assay. We investigated the discrepant observation of an undetectable viral load in an immunodeficient pregnant HIV-1-infected patient of African origin with no prior antiretroviral treatment. Although clinical progression was present in this patient with tuberculosis and a low CD4 cell count, viral load determinations with both the Amplicor Monitor and NASBA assays revealed no detectable RNA levels. The presence of HIV-1 RNA in the plasma of the patient was demonstrated by an in-house RNA-PCR. Subsequent HIV-1 RNA quantification with the branched DNA method revealed a high viremia (460,000 copies/ml). DNA sequence analysis of the gag gene identified a subtype G HIV-1 strain (HIV-1BL). To our knowledge this is the first report of a patient harboring an HIV-1 genotype of the main group with a high viral load as quantified by the branched DNA assay, but undetectable with the two commercial HIV RNA amplification techniques because of genetic divergence. In the case of discrepant low viral loads determined by one amplification technique in patients with advanced clinical stage one should use an alternative quantification technique for confirmation.

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.Hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) are major causes of mortality in both developing and developed countries. For both viruses, relatively effective therapies have been available in developed countries for quite some time. HCV and HIV-1 viral loads are important parameters in patient management both before initiating therapy and during therapy. The decline of the HCV viral load during the first 3 months of therapy is, for instance, a strong indicator of the final outcome of therapy (9, 23). Moreover, in many countries, the HCV viral load in infected health care workers determines whether they are allowed to perform surgical procedures. HIV-1 viral load is monitored during therapy, and a viral load above a certain threshold, which may differ per treating physician, requires a switch in medication (6, 13).The first tests that were described for determining the viral load were based on target amplification techniques like reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA). These tests had readout on agarose gel or readout with enzymatic detection of the amplicon after amplification with biotinylated primers (1, 3, 15, 17). Subsequently, signal amplification techniques were developed for the quantitative detection of HCV and HIV-1 (5, 16). Those techniques were improved and commercialized. The suppliers of the most widely used systems at the moment are Roche and Abbott, for RT-PCR-based systems (the COBAS Amplicor and LcX systems, respectively); bioMerieux (formerly Organon Technica), with a NASBA-based technique; and Bayer, with the Versant bDNA system being a signal amplification technique (4,18,19). Each technique has its own advantages and disadvantages. The relatively small dynamic range is a disadvantage that all current assay formats have in common. All assay formats are also rather sensitive to contamination, especially at the lower limit of detection (21). Handling of the sample after target amplification is a major cause of contamination for the NASBA-and RT-PC...