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PubMed2004

Promoter methylation status and expression of TMS1 gene in human epithelial ovarian cancer.

Gene silencing associated with aberrant DNA methylation of promoter CpG islands is one mechanism through which tumor suppressor genes are inactivated in human cancers. TMS1 (target of methylation-induced silencing) is a CpG island-associated gene that functions in the regulation of apoptosis. In this study, we examined the DNA methylation status of the TMS1 promoter in ovarian cancer cell lines and tissues by methylation-specific PCR (MSP) and its mRNA expression by reverse transcription and quantitative PCR. Aberrant methylation of TMS1 was present in 7/12 ovarian cancer cell lines and 8/20 primary ovarian cancer tissues. The median value of relative TMS1 gene expression in cancers with methylation (0.15) was significantly lower than that in cancers without methylation (13.9) (P < 0.001). The expression of the TMS1 gene was relatively high (48.5) in the normal ovarian cDNA library. TMS1 gene expression was restored by treatment with the demethylating agent 5-aza-2'-deoxycitidine in the OV90 cell line, which lacks the TMS1 transcript. Our results suggest that aberrant methylation of TMS1 may play a role in the pathogenesis of ovarian cancer.

CARD Signaling Adaptor Proteins↗
PubMed2004

Constitutive and functional expression of cyclooxygenase 2 in the murine proximal colon.

Recent reports suggest that cyclo-oxygenase (COX)-2, an inducible COX isoform may be constitutively expressed in gastrointestinal tissues. This study has evaluated the expression and function of COX-2 in the tunica muscularis of the murine proximal colon. Cyclo-oxygenase-2-like (COX-2-LI) immunoreactivity was found in a subpopulation of neurones in the myenteric and submucosal ganglia and in interstitial cells of Cajal within the muscle layers (IC-IM). Reverse transcriptase polymerase chain reaction (RT-PCR) verified expression of COX-2 in colonic muscles, and quantitative PCR demonstrated that COX-1 transcriptional expression was greater than COX-2. To test the functional significance of COX-2 expression, the effects of a COX-2 inhibitor were compared with the effects of indomethacin (COX-1/COX-2 inhibitor) on circular muscle contractions. The experiments indicate that indomethacin and the specific COX-2 inhibitor, GR253035X, increased the amplitude of phasic contractions, suggesting production of inhibitory prostaglandins tonically dampen contractile activity. The effects of indomethacin were reduced when tested on phasic contractions of muscles from COX-2 knockout mice. GR253035X did not affect contractions in muscles of COX-2 knockout animals. These studies demonstrate constitutive expression of COX-2 in the tunica muscularis of the proximal colon. The COX-2 appears to contribute a significant amount of the prostaglandins that affect the contractile behaviour of colonic muscles.

Animals↗
PubMed2002

Recurrent herpes simplex virus type 2 meningitis: a case report of Mollaret's meningitis.

It is well known that herpes simplex virus (HSV) type 2 produces acute meningitis, while HSV type 2 rarely causes recurrent meningitis (Mollaret's meningitis). We report the history of a 40-year-old patient with recurrent HSV type 2 meningitis (Mollaret's meningitis). The patient had seven episodes of meningeal symptoms within a 7-year period. In the seventh episode, HSV type 2 DNA was confirmed by nested polymerase chain reaction (PCR) with the cerebrospinal fluid (CSF). A real-time quantitative PCR study of the first CSF sample detected 2,000 copies of the HSV genome, which rapidly disappeared following treatment with acyclovir. The present case may be the first case of HSV type 2 Mollaret's meningitis to be documented in Japan. In our case, HSV serum antibody titers were at low levels during the whole course of the disease. The possible pathophysiology of this case is discussed.

Acyclovir↗
PubMed2002

[The identical effects of B7-1 and B7-2 on regulation of human IL-2 gene transcription factors NF-kappa B and AP-1].

To detect effects of B7 co-stimulation on cytokines, especially on IL-2 mRNA and transcription factors NF-kappa B and AP-1, antiB7-1 McAb, antiB7-2 McAb and C TLA-4 Ig were added into mixture lymphocyte reaction (MLR) system to block B7/C D28 signal transduction, IL-2 mRNA and IL-4 mRNA were determined by using competitive PCR and IFN-gamma mRNA by using semi-quantitative PCR. MHC class II molecules and B7 transfectants were used to stimulate CD28(+) T cell, effects of B7 on NF-kappa B and AP-1 were detected by DNA-protein binding assay. The results showed that IL-2, IL-4 and IFN-gamma mRNA were significantly lower when blockade of B7-2 in MLR than blockade of B7-1. Synergistic effects could be seen with combination of two or three antibodies. One to six hours after MLR, tDR7 alone induced NF-kappa B binding activity; cotransfecting B7 no significantly difference at early time point. After 6 hours, induction of tDR7 was decreased whereas B7 prolonged the induction of NF-kappa B till 72 hours. tDR7 alone also upregulated AP-1 binding activity, on the contrary to NF-kappa B, co-transfecting B7-1 and B7-2 decreased AP-1 binding activity within 24 hours. But during 48 - 72 hours, B7 also prolonged the AP-1 binding activity. It is concluded that after MLR, B 7 increased IL-2 secretion by decreasing the degradation of IL-2 mRNA and upregulating IL-2 transcription factors. B7 also induced several kinds of cytokines secretion. Effects of B7-1 and B7-2 had no significant difference on transcription factors. It is suggested that the different functions between B7-1 and B7-2 maybe related to the difference of cell expression and kinetics. To study the molecular mechanism of B7 mediated T cell immune tolerance can help us to design a better clinic schema to prevent transplantation rejection and GVHD.

3T3 Cells↗
PubMed2004

[Quantitative analysis of Epstein-Barr virus DNA in plasma and peripheral blood cells in patients with nasopharyngeal carcinoma].

OBJECTIVE: To quantitative analysis of Epstein-Barr virus (EBV) DNA levels in plasma, peripheral blood cells (PBCs) and tumor tissue in nasopharyngeal carcinoma (NPC), to investigate the relationship between EBV-DNA levels and clinical parameters. METHODS: Blood of 150 primary NPC and 49 corresponding tumor tissues, 47 nasopharyngitis tissues and blood of 75 controls were entered this investigation. Plasma and PBCs were isolated for quantitative detection of EBV-DNA by using real-time quantitative PCR (RQ-PCR). The paraffin-embedded tissue sections were conducted to quantitative detection of EBV-DNA, and EBER1 in situ hybridization (ISH) for calculating the percentage of positive cells on the tissue section. RESULTS: Plasma EBV-DNA levels and detecting rate in NPC before treatment (median 82 500 copies/ml, 92%) were significantly higher than that in NPC after treatment (median 0 copy/ml, 19%) and in controls (median 0 copy/ml, 12%) (P < 0.05), whereas there was no significant difference between NPC after treatment and controls (P > 0.05). There was no significant difference of PBCs EBV-DNA load and detecting rate in NPC before (0 copy/actin, 24%) and after treatment (0 copy/actin, 14%), as well as in controls (0 copy/actin, 16%) (P > 0.05). Plasma EBV-DNA level was not correlated to PBCs EBV-DNA load in NPC before (P = 0.92) and after treatment (P = 0.27), and controls (P = 0.74). EBV-DNA level (27.8 copies/actin) in NPC tumor tissues was significantly higher than that in nasopharyngitis (0 copy/actin) (P < 0.05), and was positively correlated to the ratio of EBER1 positive cells to total cells on the NPC section. In NPC patients, plasma EBV-DNA level was significantly increased in TNM stage I (2500 copies/ml), II (32 590 copies/ml), III (86 000 copies/ml) and IV (166 200 copies/ml), whereas there was no significantly difference of PBCs EBV-DNA loads in difference stages of NPC. CONCLUSION: Plasma EBV-DNA level is a more sensitive and reliable biomarker than PBCs EBV-DNA loads for reflection the tumor volumes in NPC patients. Plasma EBV-DNA detection will improve TNM staging in NPC clinical practice on molecular level.

Biomarkers, Tumor↗
PubMed2004

Update on herpes simplex encephalitis.

Herpes simplex encephalitis is the most common identified cause of sporadic viral encephalitis in the United States. Early diagnosis is critical because treatment with the antiviral drug acyclovir dramatically decreases morbidity and mortality. The use of polymerase chain reaction (PCR) techniques to amplify the genome of herpes simplex virus (HSV) from cerebrospinal fluid (CSF) has become the diagnostic procedure of choice. False-positive CSF HSV PCR results are rare when testing is performed in experienced laboratories. Negative CSF HSV PCR results should always be interpreted in the context of the timing of specimen collection and the likelihood of disease. Negative CSF HSV PCR tests can occur within the first 72 hours of illness, with subsequent tests becoming positive. Patients with HSV encephalitis will typically have a negative CSF HSV PCR after 14 days of acyclovir treatment, and a persisting positive PCR should prompt consideration of additional or revised antiviral therapy. Quantitative PCR testing provides information about HSV viral load in CSF, but the potential correlation of viral load with prognosis or other clinical features of disease remains uncertain. Although the neuroimaging abnormalities seen in HSV encephalitis are not unique, more than 90% of patients with proven HSV encephalitis will have magnetic resonance imaging (MRI) abnormalities involving the temporal lobes. Special MRI techniques, including fluid-attenuated inversion recovery and diffusion-weighted imaging, might reveal abnormalities not seen with conventional imaging sequences. Neuroimaging patterns in infants and children differ significantly from those seen in adults and include a higher frequency of extratemporal lesions.

Acyclovir↗
PubMed2005

High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer.

In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.

DNA Primers↗
PubMed2004

Real time TaqMan PCR detection and quantitation of HBV genotypes A-G with the use of an internal quantitation standard.

BACKGROUND: Diagnostic assays for the accurate quantitation of hepatitis B virus (HBV) DNA levels from patients undergoing antiviral therapy are useful for monitoring and tailoring therapy. Such assays should give accurate results with all HBV genotypes, including a seventh genotype of hepatitis B virus, genotype G, that has recently been identified in specimens from HBV-positive patients in the United States and Europe. OBJECTIVES: To characterize the performance characteristics of a quantitative real time TaqMan PCR assay, the High Pure System Viral Nucleic Acid/COBAS TaqMan HBV Test, for the detection and quantitation of HBV genotypes A-G from patient plasma and serum. This test was evaluated for limit of detection, dynamic range, reproducibility, accuracy, and genotype inclusivity. STUDY DESIGN: Primers and TaqMan probes specific for HBV and an internal quantitation standard (QS) were designed and tested using a set of plasmid DNAs representing various genotyped specimens. In addition, sensitivity, dynamic range, precision, and correlation with the COBAS AMPLICOR HBV MONITOR Test were evaluated using HBV dilution panels and patient specimens. RESULTS AND CONCLUSIONS: A real time TaqMan assay was developed that detects and quantifies DNA from genotypes A-G equivalently. The assay has a limit of detection of <50 copies/ml with plasma and serum, a dynamic range up to 10(9) copies/ml, and good precision and accuracy. Titers obtained with this method without dilutions show good correlation across the dynamic range with titers obtained with the COBAS AMPLICOR HBV MONITOR Test.

DNA Primers↗
PubMed2004

Effects of cell cycle on telomerase activity and on hepatitis B virus replication in HepG2 2.2.15 cells.

BACKGROUND: It has been shown that telomerase activity and hepatitis B virus (HBV) replication are closely associated with cell cycle. This study aimed to further investigate the effects of cell cycle on telomerase activity and on HBV replication. METHODS: Human hepatoma cells transfected with HBV DNA (HepG2 2.2.15 cell line) were treated respectively with serum deprivation, all-trans retinoic acid (RA), dimethyl sulfoxide (DMSO), or sodium butyrate. The cell cycle of HepG2 2.2.15 cells was analyzed by flow cytometry. The telomerase activities of the cells were detected by TRAP-PCR-ELISA. HBV DNA in culture medium was assayed by a fluorescent quantitative PCR assay and a semiquantitative dot blot hybridization technique. HBsAg and HBeAg in culture media were quantitatively examined by an ELISA assay. RESULTS: Treatments with serum deprivation, RA, DMSO, or sodium butyrate inhibited the proliferation of HepG2 2.2.15 cells and led to cell arrest in the G0/G1 phase of cell cycle. The percentage of the G0/G1 phase in the groups of sodium butyrate, DMSO, RA and serum-free was 85.2%, 71.9%, 68.3% and 65.2%, respectively, but in the control group, 43.1% (P<0.01). The activities of telomerase of the cells were also significantly inhibited by 82.8%, 74.6%, 76.1% and 69.4% respectively. In addition, HBV replication of the HepG2 2.2.15 cells remarkably increased as shown by the contents of HBV DNA, HBsAg and HBeAg in the culture media of the cells treated with sodium butyrate, DMSO, RA or serum deprivation (P<0.01). The amounts of HBV DNA in the groups of sodium butyrate, DMSO, RA, serum deprivation and control were 6.7X10(6), 4.8X10(6), 4.4X10(6), 5.1X10(6) and 1.2X10(6) copies/ml, respectively (P<0.01). Telomerase was expressed mainly in the cells in S phase. HBV replication increased in quiescent cells (G0/G1 phase), and decreased in proliferating phase (S phase). CONCLUSION: The current data approve that HBV replication is associated with the cellular proliferative activity.

Carcinoma, Hepatocellular↗
PubMed2003

Differential regulation of gene expression by ovariectomy in mouse aorta.

The purpose of this study was to investigate the effects of ovarian hormones on gene expression in the vascular wall. Our approach employed an RT-PCR-based cloning strategy of DNA differential display analysis and verification/confirmation of differential expression by semi-quantitative PCR and real-time PCR. mRNA analysis of normal aortas from intact and ovariectomized female C57BL/6J mice, showed altered expression of 20 genes with significant (>70%) sequence homology to known genes. Eight were selected for further study based on the genes' known function and potential relevance to vascular physiology. Differential expression of mRNA for three genes was confirmed by both semi-quantitative and real-time RT-PCR using gene-specific primers. Ovariectomy downregulated expression of elongation factor-1alpha (3.5-fold), ganglioside-induced differentiation associated protein (8.2-fold), and NADH:ubiquinone oxidoreductase (3.8-fold). Thus, in normal mouse aortas, ovariectomy resulted in significant differential downregulation of a number of vascular genes important to vascular cell growth and angiogenesis, cellular differentiation, and mitochondrial energy metabolism, respectively. These studies have implications for our understanding of hormonal regulation of vascular gene expression and the therapeutic targeting of specific vascular genetic sequences by female sex steroid hormones.

Animals↗
PubMed2002

Chemotherapy targeting methylthioadenosine phosphorylase (MTAP) deficiency in adult T cell leukemia (ATL).

Methylthioadenosine phosphorylase (MTAP) is an important enzyme used for the salvage of adenine and methionine. Cells lacking this enzyme are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation. We reported previously that the MTAP gene is deleted in adult T cell leukemia (ATL) cells. In the present study, we expanded our series and used a real-time quantitative PCR assay for accurate diagnosis of the deletion and nine of 65 primary ATL samples (13.8%) were MTAP negative. In spite of this low incidence, ATL cells showed significantly higher sensitivity to L-alanosine, an inhibitor of de novo adenosine monophosphate (AMP) synthesis, than normal lymphocytes, suggesting that the MTAP gene is inactivated not only by deletion but also by other mechanisms. Indeed, a real-time quantitative RT-PCR assay disclosed that primary ATL cells had significantly lower MTAP mRNA expression than normal lymphocytes. Since MTAP-negative ATL cell lines also showed much higher sensitivity to L-alanosine than MTAP-positive ATL cell lines, we used these cell lines to investigate whether it is possible to develop selective therapy targeting MTAP deficiency. A substrate of MTAP, methylthioadenosine (MTA) or its substitutes rescued concanavalin A (Con A)-activated normal lymphocyte proliferation from L-alanosine toxicity. All the compounds except 5'-deoxyadenosine, however, also caused the undesirable rescue of MTAP-negative ATL cell lines. 5'-Deoxyadenosine had the desired ability to rescue hematopoietic progenitor cells without rescuing ATL cell lines. These results support the rationale for a chemotherapy regimen of L-alanosine combined with 5'-deoxyadenosine rescue in MTAP-deficient ATL.

Adenosine Monophosphate↗
PubMed2006

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

Animals↗
PubMed2004

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas).

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.

Animals↗
PubMed2005

Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping.

High-resolution melting of polymerase chain reaction (PCR) products can detect heterozygous mutations and most homozygous mutations without electrophoretic or chromatographic separations. However, some homozygous single nucleotide polymorphism (SNPs) have melting curves identical to that of the wild-type, as predicted by nearest neighbor thermodynamic models. In these cases, if DNA of a known reference genotype is added to each unknown before PCR, quantitative heteroduplex analysis can differentiate heterozygous, homozygous, and wild-type genotypes if the fraction of reference DNA is chosen carefully. Theoretical calculations suggest that melting curve separation is proportional to heteroduplex content difference and that the addition of reference homozygous DNA at one seventh of total DNA results in the best discrimination between the three genotypes of biallelic SNPs. This theory was verified experimentally by quantitative analysis of both high-resolution melting and temperature-gradient capillary electrophoresis data. Reference genotype proportions other than one seventh of total DNA were suboptimal and failed to distinguish some genotypes. Optimal mixing before PCR followed by high-resolution melting analysis permits genotyping of all SNPs with a single closed-tube analysis.

Electrophoresis, Capillary↗
PubMed2004

Adaptation of PCR technique for quantitative estimation of genetic material from different regions of chromosome 21 in cases of trisomy 21.

Pre- and postnatal diagnosis of chromosomal aberrations is generally based on conventional cytogenetic analysis. In this paper, we have devised a quantitative polymerase chain reaction (Q-PCR) method to determine gene dose effects and applied it in cases of regular trisomy 21 as a model. The method is based on quantitative assessment of PCR products after using primers amplifying DNA fragments located in the pericentromeric, heterochromatic, euchromatic and telomeric regions of chromosome 21. A gene dose effect on the amount of PCR product in cases of trisomy 21 was confirmed. Moreover, a correlation between the amount of the PCR product of the examined sequences and their location in the chromosome was observed. The obtained results suggest that the Q-PCR technique can be applied in the diagnosis of aneuploidies.

Amyloid beta-Protein Precursor↗
PubMed2006

Caveolin-1 and -2 in airway epithelium: expression and in situ association as detected by FRET-CLSM.

BACKGROUND: Caveolae are involved in diverse cellular functions such as signal transduction, cholesterol homeostasis, endo- and transcytosis, and also may serve as entry sites for microorganisms. Hence, their occurrence in epithelium of the airways might be expected but, nonetheless, has not yet been examined. METHODS: Western blotting, real-time quantitative PCR analysis of abraded tracheal epithelium and laser-assisted microdissection combined with subsequent mRNA analysis were used to examine the expression of cav-1 and cav-2, two major caveolar coat proteins, in rat tracheal epithelium. Fluorescence immunohistochemistry was performed to locate caveolae and cav-1 and -2 in the airway epithelium of rats, mice and humans. Electron-microscopic analysis was used for the identification of caveolae. CLSM-FRET analysis determined the interaction of cav-1alpha and cav-2 in situ. RESULTS: Western blotting and laser-assisted microdissection identified protein and transcripts, respectively, of cav-1 and cav-2 in airway epithelium. Real-time quantitative RT-PCR analysis of abraded tracheal epithelium revealed a higher expression of cav-2 than of cav-1. Immunoreactivities for cav-1 and for cav-2 were co-localized in the cell membrane of the basal cells and basolaterally in the ciliated epithelial cells of large airways of rat and human. However, no labeling for cav-1 or cav-2 was observed in the epithelial cells of small bronchi. Using conventional double-labeling indirect immunofluorescence combined with CLSM-FRET analysis, we detected an association of cav-1alpha and -2 in epithelial cells. The presence of caveolae was confirmed by electron microscopy. In contrast to human and rat, cav-1-immunoreactivity and caveolae were confined to basal cells in mice. Epithelial caveolae were absent in cav-1-deficient mice, implicating a requirement of this caveolar protein in epithelial caveolae formation. CONCLUSION: These results show that caveolae and caveolins are integral membrane components in basal and ciliated epithelial cells, indicating a crucial role in these cell types. In addition to their physiological role, they may be involved in airway infection.

Animals↗
PubMed2006

Comparison of two commercially available pp65 antigenemia tests and COBAS Amplicor CMV Monitor for early detection and quantification of episodes of human CMV-viremia in transplant recipients.

The performance of two commercially available CMV pp65 antigenemia (AG) assays (CINA kit, Argene Biosoft, CK, and CMV Brite kit, IQ Products, CBK), and a quantitative PCR test (COBAS Amplicor CMV Monitor Test, Roche Diagnostics, CACM) was evaluated with 667 blood samples from 215 transplant recipients. The diagnostic value of the three tests was defined by their ability to detect episodes of systemic CMV activity. A test score was defined, assigning 7 virtual units (VU) for any episode detected first, 5 VU for any episode detected second, and three VU for any episode detected last. Within the evaluated collective, the following overall scores could be determined for the three assays: 45 VU for the CK, 39 for CACM and 31 for the CBK assay. We conclude from our results that (1) the CK is superior to the CBK assay in detecting episodes of CMV antigenemia, and (2) quantitative serum CMV PCR with CACM is not generally superior to pp65 antigenemia testing.

Antigens, Viral↗
PubMed2001

Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes.

The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real-time quantitative PCR test based on fluorescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based on the detection of 10 pg mouse DNA added to the AF was included to check for PCR efficiency. The relationship between the parasite load and the occurrence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Seven AF (8%) had a parasitic load > 10(3); 14 (16%) had > 10(2)-< or =10(3); 26 (30%) had > 10-< or = 10(2); and 40 (46%) had < or = 10 parasites/ml. Four of the six AF with cerebral ventriculomegaly had >10(3) parasites/ml. The other two had 130 and 24 parasites/ml, respectively. No parasitic loads of > 10(3) parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to associate high parasite count with ultrasonographic abnormality, but the main concern remains early maternal infection. The importance of quantification should be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results.

Amniocentesis↗