Hongxin Fan

Epstein-Barr Virus Quantitation by Real-Time PCR Targeting Multiple Gene Segments: A Novel Approach to Screen for the Virus in Paraffin-Embedded Tissue and Plasma

Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.

Epstein-Barr Virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies.

Epstein-Barr Virus (EBV), a ubiquitous gamma herpes virus, infects more than 95% of the human population before adulthood. Life-long persistence, usually without adverse health consequences, relies on a balance between viral latency, viral replication, and host immune response. Patients with EBV-related disease often have high levels of EBV DNA in their plasma. This study addresses whether this circulating, cell-free EBV DNA is encapsidated in virions or exists as naked genomes. First, an assay was developed, combining DNase I and quantitative real-time PCR, to discriminate encapsidated from naked EBV DNA. EBV DNA was almost always naked in the plasma of AIDS-related lymphoma patients (n = 11) and immunosuppressed/posttransplantation patients (n = 8). In contrast, infectious mononucleosis patients (n = 30) often had a mixture of encapsidated and naked EBV DNA. These findings may be important in understanding how viral load relates to disease status and in predicting response to nucleoside analogs and other antiviral therapies.

Laboratory markers of tumor burden in nasopharyngeal carcinoma: A comparison of viral load and serologic tests for Epstein-Barr virus

Epstein‐Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real‐time or end‐point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non‐NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end‐point and real‐time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.

First Detection of TR34/L98H and TR46/Y121F/T289A Cyp51 Mutations in Aspergillus fumigatus isolates in the United States

ABSTRACT Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here.

Molecular methods for detecting t(11;14) translocations in mantle-cell lymphomas

The t(11;14)(q13;q32) and its molecular counterpart, bcll/JH, are characteristic of mantle-cell lymphomas (MCL). Molecular detection of the translocation is useful in diagnosis and classification, and also shows promise in detecting minimal residual disease. The purpose of this study was to determine the frequency of detecting bcll/JH by polymerase chain reaction (PCR) compared with Southern blot analysis in cases proven by cytogenetic analysis to harbor t(11;14). Southern blot analysis using two probes targeting the major translocation cluster (MTC) and a third probe targeting the p94 region was performed, along with PCR using two different bcll MTC primers, on 18 cases of MCL known to have t(11;14). Southern blot analysis revealed bcll rearrangement in 13 of 18 cases (72%), 12 with MTC breakpoints and 1 with a p94 breakpoint. The 2.1-kb MTC probe “b” was superior to the smaller 700-bp probe “a” in detecting these rearrangements. The MTC translocation was identified by PCR in 10 of 12 cases, and both primer sets that were tested performed equally well. This study illustrates the frequency with which molecular methods detect known t(11;14) translocations in MCLs. These results may help clinical laboratory scientists optimize their procedure for detecting bcll translocations by molecular methods at initial diagnosis and for purposes of detecting minimal residual disease.

Quantitation of CMV by real‐time PCR in transfusable RBC units

BACKGROUND: CMV is one of the most significant pathogens infecting immunocompromised individuals. CMV is transmissible through transfusion of blood components. The goal of this study was to measure CMV levels in RBC units using a sensitive and quantitative DNA amplification assay.

Mosaicism for an FMR1 gene deletion in a fragile X female

Most cases of fragile X syndrome result from expansion of CGG repeats in the FMR1 gene; deletions and point mutations of FMR1 are much less common. Mosaicism for an FMR1 full mutation with a deletion or with a normal allele has been reported in fragile X males. Here we report on a fragile X female who is mosaic for an FMR1 full mutation and an intragenic deletion. The patient is a 4‐year‐old girl with developmental delay, autistic‐like behaviors, and significant speech and language abnormalities. Southern blotting demonstrated the presence of a methylated full mutation, a normal allele in methylated and unmethylated forms, and an additional fragment smaller than the normal methylated allele. This result indicates that the patient is mosaic for a full mutation and a deletion, in the presence of a normal allele. By DNA sequence analysis, we mapped the 5′ breakpoint 63/65 bp upstream from the CGG repeat region and the 3′ breakpoint 86/88 bp downstream of the CGG repeats within the FMR1 gene. The deletion removed 210 bp, including the entire CGG repeat region. The full mutation was inherited from a premutation in the patients mother. The deletion, which remained methylated at the Eag I and Nru I sites, was probably derived from the full mutation allele. Mosaicism of this type is rare in females with a fragile X mutation but should be kept in mind in the interpretation of Southern blots.

Cytomegalovirus DNA measurement in blood and plasma using Roche LightCycler CMV quantification reagents.

Cytomegalovirus (CMV) is a common cause of morbidity and mortality in immunosuppressed patients. Newly available commercial systems facilitate the measurement of CMV DNA in whole blood or plasma, as a means of detecting and monitoring active disease. We evaluated the performance characteristics of a quantitative polymerase chain reaction that relies on analyte-specific reagents and instruments from Roche Diagnostics. DNA was extracted using a MagNaPure instrument from blood and matched plasma specimens of patients with active CMV disease (defined by another molecular assay) and from controls. Viral load was measured against Roches standards on a LightCycler instrument, followed by melt curve analysis to confirm product specificity. Dual hybridization probes targeted the CMV UL54 gene and a control sequence that was spiked before extraction. Accuracy and linearity were established using spiked DNA from the Towne-strain CMV. The assay was linear across 6 logs, and it detected CMV DNA in 67/70 blood samples (96%) from patients who were considered to have an active CMV infection, including all 67 viral loads above 208 CMV copies/mL, suggesting that it was sensitive enough to detect clinically significant infections in immunosuppressed patients. Virus levels in plasma correlated reasonably well with the levels in whole blood (r2=0.5756), suggesting that levels in either plasma or blood level were indicative of active infection. It was important to verify the calculated values by visualizing the amplification plot and using the melt curve analysis to resolve discrepancies. The LightCycler CMV assay is rapid, sensitive, and linear for quantifying CMV viral load, and it seems to be useful in the diagnosis and monitoring of affected patients.

Lymphoid Tissues from Patients with Infectious Mononucleosis Lack Monoclonal B and T Cells

In typical cases of infectious mononucleosis (IM), lymphoid tissue is rarely submitted for pathological examination. When lymphoid tissues from IM cases are examined, the histological appearance of IM may be difficult to distinguish from malignant lymphoma. The purpose of this study was to address the utility of clinical molecular assays for T and B cell clonality in distinguishing IM from lymphoid malignancy. DNA was recovered from paraffin-embedded archival lymphoid tissues of 18 cases of IM and 13 control cases representing other reactive lymphoid hyperplasias. T cell receptor gamma (TCR-gamma) and immunoglobulin heavy chain (IgH) gene rearrangements were assayed using our standard clinical polymerase chain reaction procedures targeting each of the four functional variable (V) families and the three joining (J) families of the TCR-gamma gene, and framework III of the IgH gene, respectively. In 17 of 18 cases of IM, no monoclonal T or B cell populations were detectable. One case, the only spleen specimen in the study, had an oligoclonal pattern of TCR-gamma rearrangements. The control cases representing other reactive hyperplasias also lacked monoclonality. The assays used were sensitive to clonal populations as small as 5% of cells. In this case series, no monoclonal lymphoid populations were identified in any case of IM. This finding suggests that molecular studies are useful in distinguishing IM from lymphoid neoplasms.

Analytic validation of a quantitative real-time PCR assay to measure CMV viral load in whole blood

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in immunocompromised patients. We compared the CMV pp65 antigenemia test with a less labor intensive quantitative polymerase chain reaction (PCR) assay in 109 whole blood samples predominantly from transplant patients and patients with AIDS. DNA was amplified on an Applied Biosystems 7900 instrument using a TaqMan probe targeting the CMV polymerase gene and the APOB human control gene. The DNA assay was linear over a 6-log range from 8 to 800,000 CMV genomes per reaction; coefficient of variation was 20%. CMV DNA was undetectable in 20 blood samples from healthy donors whereas it was detected in 55 of 109 patient samples. Results were concordant in a nonlinear fashion with those of the antigenemia test in 90/109 (83%). Evaluation of the discrepancies suggested that either PCR or antigenemia assays could be falsely negative when virus levels were quite low. A point mutation interfered with probe binding in 1 sample. A second real-time PCR targeting the immediate early gene was even more likely to be false negative. In summary, CMV viral load measurement targeting the polymerase gene is nearly equivalent to the antigenemia assay for detecting and monitoring active CMV infection in whole blood samples.