Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina

BACKGROUND: Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection. METHODS: The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology. RESULTS: The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive. The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates. Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found. Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative. CONCLUSIONS: Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay

Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients▿

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting

A proposal for a common nomenclature for viral clades that form the species varicella-zoster virus: summary of VZV Nomenclature Meeting 2008, Barts and the London School of Medicine and Dentistry, 24–25 July 2008

Varicella-zoster virus (VZV), the cause of chickenpox and zoster, was the first human herpesvirus to be sequenced fully and the first for which vaccines have been licensed and widely used. Three groups have published genotyping schemes based on single nucleotide polymorphisms (SNPs) and, between them, have identified five distinct phylogenetic clades, with an additional two putative clades. Sequencing of over 23 whole VZV genomes from around the world further refined the phylogenetic distinctions between SNP genotypes. Widespread surveillance in countries in which the varicella vaccine is now in use and the difficulties posed by three unique genotyping approaches prompted an international meeting, at which a common nomenclature based on phylogenetic clades was agreed upon. In this paper, we review the original genotyping schemes and discuss the basis for a novel common nomenclature for VZV strains. We propose a minimum set of SNPs that we recommend should be used to genotype these viruses. Finally, we suggest criteria by which novel clades can be recognized

Early diagnosis of cytomegalovirus infection using monoclonal antibodies and DNA amplification

Human cytomegalovirus (HCMV) is ubiquitous and frequently infects normal individuals with few serious clinical consequences. However, patients with impaired cellular immunity frequently develop life-threatening opportunistic HCMV infections due to either primary infection or reactivation of latent virus. Pneumonia is the most common manifestation of HCMV visceral organ disease in solid organ and bonemarrow transplant recipients while retinitis is the most common manifestation in AlDS patients. Exposure of the fetus to HCMV in utero may lead to multi- organ system damage and permanent neurological sequelae. Early intervention with antiviral drugs depends on early sensitive and specific virological diagnosis. Detection of anti-CMV antibodies and virus isolation do not allow an early and presymptomatic diagnosis of HCMV infection. The work described has focused on the development of methods to enable the rapid and simple detection of CMV immediate early (IE), early (E) and structural CMV proteins or the detection of amplified CMV DNA in urine, bronchoalveolar lavage (BAL) or blood samples obtained from immunocompetent or immunosuppressed patients. CMV mouse monoclonal antibodies were produced and three of them, (DDG9, CCH2 and AAC 10) were further characterized and used in the different assays. Urine samples from children attending day care centres and from kidney transplant recipients were used for the development of a new method for the detection of early CMV antigen in infected cell cultures. IF staining with the CCH2 monoclonal antibody at 24 hours post-inoculation using urine from kidney transplant recipients revealed a sensitivity of 70% compared to virus isolation. The sensitivity was increased to 85% by the use of biotin- streptavidin and a new immunoenzymatic staining (IENZ) procedure. However, viremia is a better marker for disease development and further studies concentrated on the detection of CMV in blood. The CMV antigenemia assay, which can quantitatively detect CMV pp65 antigen in leukocytes during CMV infection, and a nested polymerase chain reaction (PCR) for amplification of CMV DNA from plasma were established and evaluated. We found plasma-PCR and the CMV antigenemia assay to be almost equally sensitive. However, CMV antigenemia gave the highest positive predictor value (ppv) (57%), for the development of symptomatic CMV infection in kidney transplant recipients, compared to 49% ppv for CMV DNA in plasma. In imrnunosuppressed patients with symptoms of pulmonary infection, amplification of CMV DNA by PCR from bronchoalveolar lavage (BAL) fluids was the most sensitive method for detection of CMV. The sensitivity was lower but the specificity was higher for the demonstration CMV E antigen and E antigen in infected cell cultures and for the direct detection of CMV antigens in BAL cells. For rapid diagnosis, a combination of these three methods are recommended. A negative PCR result had a 100% negative predictive value for the development of CMV pneumonia for the following two months. Three CMV gene regions, (major immediate early (ME), DNA polymerase, and glycoprotein B (gB)) were sequenced for 46 isolates obtained from four patient populations in order to assess the nucleotide stability and to evaluate the suitability of the three gene regions as targets for CMV DNA amplification. In diagnostic PCR assays, most laboratories use primers from the MIE gene region. However, we found the MIE to be the most variable gene region, indicating that this gene region is less suitable for diagnostic PCR primer selection compared to the more conserved DNA polymerase and gB gene regions. Our studies have contributed to the development of new, more optimal diagnostic methods for early diagnosis of CMV infection. The monoclonal antibodies have proved to be powerful tools for the detection of CMV in infected cells. With the new sequence data, new more optimal primer sequences can be selected and used in diagnostic PCR assays in order to detect the majority of CMV strains circulating in society. Key words: cytomegalovirus, monoclonal antibodies, polymerase chain reaction, CMV early antigen, CMV pp65 antigen. ISBN 91-628-2321-

Evaluation of the ReSSQ Assay in Relation to the COBAS AMPLICOR CMV MONITOR Test and an In-House Nested PCR Method for Detection of Cytomegalovirus DNA

The ReSSQ CMV assay is a novel commercially available kit for quantification of cytomegalovirus (CMV), based on real-time PCR with a peptide nucleic acid probe coupled with a single dye. In combination with the LightCycler, the ReSSQ CMV assay was evaluated with respect to specificity, PCR inhibition, linearity, reproducibility, and sensitivity. All nontested CMV materials were negative, and the assay was not inhibited by the use of different anticoagulants or other factors that may influence blood samples. The dynamic range was between 10 and 5 × 10(8) copies/PCR, and intra- and interassay variabilities were below 0.10 and 0.12 log(10) standard deviations, respectively. Assay sensitivity was validated by analysis of 24 samples from a proficiency panel and by comparison to a nested in-house CMV PCR and the COBAS AMPLICOR CMV MONITOR test, using 159 clinical samples. Results from the proficiency panel were well in accordance with input values over the entire range of viral concentrations tested (50 to 31,250 copies/ml). The association between the ReSSQ CMV assay and the in-house PCR was in agreement in 90% of the clinical samples, and discordant results were found for all types of sample materials analyzed. The ReSSQ CMV and COBAS AMPLICOR assays showed no significant differences for samples containing >1,000 CMV copies/ml, but results differed to a greater extent at lower viral concentrations. The results demonstrate that the ReSSQ CMV assay is a CMV-specific, robust, and reproducible method and hence is well suited for routine use in clinical virology laboratories