A regimen combining the Wee1 inhibitor AZD1775 with HDAC inhibitors targets human acute myeloid leukemia cells harboring various genetic mutations.

AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

Clinical utility of a quantitative polymerase chain reaction for diagnosis of cytomegalovirus disease in solid organ transplant patients.

BACKGROUND: Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in solid organ transplant patients remains a challenge. We evaluated the clinical utility of a quantitative polymerase chain reaction (QPCR) method to diagnose transplant patients with HCMV disease. METHODS: A total of 429 plasma samples from 121 solid organ transplant patients were prospectively collected and evaluated for HCMV using a QPCR assay. To enhance the sensitivity of the QPCR assay, plasma samples were centrifuged in a manner designed to concentrate the virions before nucleic acid extraction. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) that contained the same primer sequences as for HCMV. Polymerase chain reaction products were detected by hybridization to 96-well microtiter plates coated with either a HCMV- or an IS-specific probe. RESULTS: A total of 103 patients had all samples negative by QPCR. None of the 103 patients developed HCMV disease during the study. In contrast, 18 patients showed at least 1 sample positive by the QPCR assay, but only 8 of these developed HCMV disease. The mean viral load value for patients without HCMV disease was 93 viral copies (vc) per ml of plasma (range: 35-325 vc/ml plasma) and for the 8 patients with HCMV disease was 67,686 vc/ml plasma (range: 167-1,325,000 vc/ml plasma) (P<0.001). Using a cut-off value of 100 vc/ml plasma and clinical diagnosis of HCMV disease, the QPCR assay showed a sensitivity of 100% and specificity of 99.1%. CONCLUSION: HCMV viral load may be useful in the diagnosis of HCMV disease in solid organ transplant patients.

Utility of cytomegalovirus viral load in renal transplant patients in Argentina.

BACKGROUND: Cytomegalovirus (CMV) is the most prevalent viral disease in solid organ transplantation. Detection of CMV DNA in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) frequently occurs in renal allograft recipients, yielding false positive results in seropositive patients free of CMV disease. We evaluated the clinical utility of a quantitative PCR-enzyme-linked immunosorbent assay (ELISA) for identifying patients with CMV disease. METHODS: Three hundred and fifty samples from 65 consecutive renal transplant recipients were studied. DNA was extracted from PBMC weekly up to the day of discharge and after any further admission. Samples were tested by a qualitative PCR method, and all positive samples were further studied by a quantitative PCR-ELISA method. The quantitative PCR-ELISA method used an internal standard (IS) that contained the primer sequences used in the qualitative CMV PCR. Detection and quantification was performed in 96-well plates coated with IS or CMV specific probes. RESULTS: Forty-one of 65 patients (63.1%) showed positive results by the qualitative PCR, but only 8 of these patients were diagnosed with CMV disease. Positive samples were re-analyzed by the quantitative assay. The 8 patients with CMV disease had a mean CMV viral load of 1,438+/-687 viral copies (VC)/10(6) PBMC, and the 33 without CMV disease had a mean value of 219.6+/-117.2 VC/10(6) PBMC (P<0.01). None of the 33 patients without CMV disease had viral loads higher than 500 VC/10(6) PBMC. Using 500 VC/10(6) PBMC as a cut-off value for CMV disease, the quantitative PCR showed a sensitivity and specificity of 100% compare to clinical diagnosis. CONCLUSION: CMV viral load may be useful in the diagnosis of CMV disease in renal transplant patients.

Fluorescence in situ hybridization detectable mosaicism for Angelman syndrome with biparental methylation.

We present a child with mild to moderate global developmental delay including severe speech impairment, inappropriate happy demeanor, wide-based gait, frequent ear infections with mild hearing loss, deep-set eyes, a wide mouth, widely-spaced teeth, normal head circumference, and no seizures. Results of peripheral blood lymphocyte chromosomal analysis with GTG banding were normal. However, fluorescence in situ hybridization (FISH) studies showed mosaicism for a deletion of probes (D15S10 and SNRPN) from the Angelman syndrome (AS) critical region with approximately 40% of peripheral lymphocytes having the deletion. The deleted chromosome 15 also showed centromeric duplication, which was detected with a D15Z1 probe [46,XX, dic(15)(pter-->q11.1::p11.2-->q11. 1::q13-->qter)]. The same duplication pattern was observed in 30% of the nuclei obtained from a buccal smear. Methylation studies using polymerase chain reaction with sodium bisulfite-treated DNA demonstrated a normal biparental methylation pattern. To the best of our knowledge, this is the first case with AS and a FISH detectable deletion in a mosaic pattern. We recommend FISH studies for the detection of mosaicism in the patients with AS clinical findings even if results of the methylation studies are normal.

Pitfalls in establishing a molecular diagnostic laboratory.

Diseases are increasingly being defined in terms of genetic alterations. A body of information termed "molecular pathogenesis" is evolving which provides a framework for integrating the rapidly accumulating genetic information with the related disease process. Molecular methods with their increased sensitivity and specificity are required to gather this information. A major challenge now lies in the transfer of this molecular technology from a research environment to the clinical testing arena. Technical issues, patented technology, special facilities, personnel, and regulatories issues imposed by CLIA'88, require those desiring to perform molecular tests to pay special attention to laboratory design, personnel training, and test menu development. Although establishing a successful molecular diagnostics laboratory is a complex and difficult task, the added value of these tests can have a tremendous impact in disease diagnosis and patient management.

Detection of a novel truncated WT1 transcript in human neoplasia.

BACKGROUND: The Wilms' tumor 1 (WT1) gene encodes a transcription factor critical in urogenital development. Using a new model of prostate cancer progression that permits comparison of the cellular and molecular properties of increasingly aggressive sublines of simian virus 40 large T-antigen-immortalized human prostate epithelial cells within the same lineage, the role of WT1 in tumorigenesis was investigated. METHODS AND RESULTS: Using RT-PCR and northern blotting, we identified a novel truncated WT1 transcript in these prostate cancer cell lines. This 2.1-kb transcript consisted of the coding region of the zinc-finger domain of WT1, together with a portion of intron 5 at the 5' end of the transcript. Furthermore, two peptides were detected by western blotting using antibodies to epitopes of the COOH terminus of WT1. Using RT-PCR, the 2.1-kb transcript was also detected in leukemia cell line K562, breast cancer cell line MCF7, and blood samples from patients with acute leukemia. CONCLUSION: These novel findings in both cell lines and patient-derived specimens suggest this new WT1 gene alteration has a potential role in the development of new diagnostic assays for some human malignancies.

Assessing Clinical Utility of Hepatitis C Virus Quantitative RT-PCR Data: Implications for Identification of Responders Among alpha-Interferon-treated Patients.

Background: Detection of hepatitis C virus (HCV) RNA in serum or plasma is currently the best means of identifying active HCV infection. In this study, we assessed the clinical utility of the HCV Amplicor Monitor (Roche Molecular Diagnostics, Branchburg, NJ) quantitative assay for monitoring viral burden and its implications for identifying responders among alpha-interferon-treated patients with chronic hepatitis C. Methods and Results: Precision and linearity were determined on aliquots of a pooled control serum. Error of the mean was normally distributed. The coefficient of variation of log10-transformed titers was 2%-6% over a range of 1.5 x 10(4)-1.5 x 10(5) copies/mL. Linearity over this range was high (R=.98-.99). Accuracy, as evaluated by comparison of split samples, showed that the Amplicor assay provided an unbiased estimate of the values from a reference laboratory. In a sample of 36 patients treated with alpha-interferon for chronic hepatitis C disease, mean viral titer declined with improvement of disease. The assay demonstrated heterogeneity among clinical responders with regard to their ability to actually clear their viral burden. Conclusions: Decreased viral burden as measured by the HCV Amplicor assay is potentially useful for monitoring individuals with HCV infection.

Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.

BACKGROUND: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. METHODS AND RESULTS: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. CONCLUSION: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.

Genetics of colorectal and breast cancer.

Colorectal and breast cancers account for a significant number of deaths due to malignant neoplasia. Laboratory medicine plays an important role in the diagnosis and management of these tumors through the application of histopathology, immunohistochemistry, and serologic identification of tumor markers. Approximately 5% to 10% of colorectal and breast cancers result from an inherited predisposition. The genes responsible for most genetically transmitted cancers have been identified, and the application of findings from molecular pathology are being evaluated. This article reviews the genetic changes that occur as a result of somatic mutation and inherited or germline mutations.

Can polymerase chain reaction help distinguish benign from malignant lymphoid aggregates in bone marrow aspirates?

OBJECTIVE: Although morphologic and immunologic clues are helpful in distinguishing benign from malignant lymphoid aggregates in bone marrow biopsies, there remain some cases in which it is not possible to arrive at a definitive diagnosis. Since the malignant aggregates are monoclonal B-cell proliferations, we sought to determine whether performing polymerase chain reaction for the immunoglobulin heavy-chain locus would be helpful in distinguishing these 2 entities. METHODS AND RESULTS: Scrapings from unstained bone marrow aspirate smears or touch preparations of bone marrow biopsies from 15 patients with benign bone marrow lymphoid aggregates and 18 patients with malignant lymphoid infiltrates were analyzed for rearrangements of the FR3 region of the immunoglobulin heavy-chain gene locus by a heminested polymerase chain reaction procedure. All specimens had amplifiable DNA, as shown by amplification of the ras proto-oncogene. None of the 15 cases of benign bone marrow lymphoid aggregates demonstrated clonality upon amplification of the immunoglobulin heavy-chain gene locus. In contrast, 8 of the 18 malignant samples were positive (P =.01 by chi(2) test; sensitivity, 44%; specificity, 100%; positive predictive value, 100%; negative predictive value, 60%). There was a tendency for there to be more lymphocytes in stained bone marrow aspirate smears from the cases of malignant lymphoid aggregates with a positive polymerase chain reaction result than in those without demonstrable clonality (36.0 +/- 35.4% vs 9.8 +/- 8.0%, P =.13). CONCLUSIONS: Polymerase chain reaction for the immunoglobulin heavy-chain gene locus may help distinguish benign from malignant bone marrow lymphoid aggregates. Although the presence of false-negative samples may be related to the relative lack of lymphocytes in the bone marrow aspirates, other factors, such as the lack of amplification of the FR3 region of the immunoglobulin heavy-chain gene locus in particular tumors, cannot be ruled out with certainty.

Primary T-cell-rich B-cell lymphoma masquerading as a meningioma.

Primary dural lymphoma is rare, and few of the small number of cases reported to date have been classified using immunohistochemical techniques. To our knowledge, we report the first case of T-cell-rich B-cell lymphoma (diffuse mixed small cell and large cell) presenting as a solitary intracranial dural mass. Cytologic and frozen sections prepared during intraoperative consultation revealed a polymorphic population of lymphocytes suspicious for an inflammatory process. Permanent sections of the dura showed a diffusely infiltrating mass composed of mature lymphocytes peppered with large atypical lymphocytes. Immunohistochemical stains identified the small lymphocytes as T cells (CD3 and CD43) and the large atypical lymphocytes as B cells (CD20). Evidence of rearranged immunoglobulin heavy-chain genes demonstrated B-cell monoclonality. Differentiating between inflammatory and neoplastic lymphocytic masses of the dura obviously has important therapeutic and prognostic significance and may require immunohistochemical and molecular techniques.

A disattenuated correlation estimate when variables are measured with error: illustration estimating cross-platform correlations.

Previous cross-platform reproducibility studies have compared consistency of intensities as well as consistency of fold changes across different platforms using Pearson's correlation coefficient. In this study, we propose the use of measurement error models for estimating gene-specific correlations. Additionally, gene-specific reliability estimates are shown to be useful in prioritizing clones for sequence verification rather than selecting clones using a simple random sample. The proposed 'disattenuated' correlation may prove useful in a wide variety of studies when both X and Y are measured with error, such as in confirmation studies of microarray gene expression values, wherein more reliable laboratory assays such as real-time polymerase chain reaction are used.

Multilaboratory comparison of hepatitis C virus viral load assays.

We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays enable them to be used for HCV diagnostics and therapeutic monitoring. However, the differences in the viral load values obtained with the different assays underscore the importance of using one assay when monitoring response to therapy.

Overexpression of C-NEU and C-MET during rat liver cholangiocarcinogenesis: A link between biliary intestinal metaplasia and mucin-producing cholangiocarcinoma.

Based on limited but compelling immunohistochemical data demonstrating individual overexpression of the tyrosine kinase growth factor receptors, c-erbB-2 and c-met, in significant percentages of human cholangiocarcinoma (ChC), we investigated if combined overexpression of both c-neu, the rat homologue of c-erbB-2, and c-met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), might represent a characteristic, early event associated with furan-induced cholangiocarcinogenesis in rat liver. Specifically, through the use of immunohistochemistry, in situ hybridization (ISH), and Western and Northern blotting, we found that both c-neu and c-met are prominently overexpressed in intestinal metaplastic lesions in early putative precancerous cholangiofibrotic tissue formed in the livers of rats after 6 weeks of furan treatment when compared with normal and hyperplastic intrahepatic biliary epithelia. We further demonstrated that c-neu and c-met are concordantly overexpressed in neoplastic glandular epithelia in later-developed primary "intestinal-type" of ChC formed in the livers of furan-treated rats, as well as in subsequently derived transplantable mucin-producing tumors. Overexpression of c-neu and c-met correlated with increased proliferating cell nuclear antigen (PCNA)-labeling indices, which were determined to be three to four times higher in intestinal metaplastic glands in precancerous cholangiofibrotic tissue and in neoplastic glands in the primary "intestinal type" of ChC than in hyperplastic bile ductular structures within either cholangiofibrotic or bile duct-ligated (BDL) livers. The c-neu and c-met receptor proteins overexpressed in different in vivo passages of a transplantable ChC each contained immunoreactive phosphotyrosines, indicating an activated state. However, we did not detect evidence of either gene amplification of c-neu or c-met or of a common transmembrane-activating mutation in c-neu expressed in transplantable ChC. Our findings indicate that altered expression of c-neu and c-met occurs relatively early in the process of furan-induced cholangiocarcinogenesis in rat liver and may play a potentially important role in its pathogenesis. They further indicate a common alteration in tyrosine kinase growth factor receptor expression linking early putative precancerous intestinal metaplastic lesions in liver to later-developed mucin-producing biliary cancer.

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids.

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two major by-products formed during chlorine disinfection of drinking water, increase the incidence of tumors in B6C3F1 mice by 6- and 3-fold respectively. In order to understand better the mechanism by which these two compounds induce liver tumors, the incidence and spectrum of mutations in the K- and H-ras proto-oncogenes in these tumors were analyzed. DNA from spontaneous, DCA- and TCA-induced liver tumor from B6C3F1 male mice was evaluated for point mutations in exons 1, 2 and 3 of the two genes by single-stranded conformation polymorphism. Results demonstrated a similar incidence of mutations for exon 2 of H-ras in spontaneous carcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%), 1.0 g/l (48%) and TCA 4.5 g/l (45%). Only four showed mutations in the other exons of Hras or in K-ras. Sequence analysis of spontaneous tumor samples with second exon H-ras mutations revealed a change in codon 61 from CAA to AAA in 80% and CAA to CGA in 20% of tumors. In contrast, tumors with H-ras mutations from DCA-treated mice revealed a H-61 change from CAA to AAA in 21% at 3.5 g/l and 16% at 1.0 g/l. CAA to CGA was observed in 50% of tumors from mice given DCA 3.5 or 1.0 g/l, and CAA to CTA was present in 29% and 34% of the two dosage groups respectively. Interestingly, TCA showed the same mutational spectrum as the spontaneous liver tumors. The data indicates that induction of liver carcinoma by DCA and TCA involves activation of the H-ras proto-oncogene at a frequency similar to that observed in spontaneous tumors. However, the mechanism(s) for including hepatocellular carcinoma does not appear to be identical for DCA and TCA.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay.

Genetic changes in solid tumors.

Although most solid tumors are treated surgically, determining the genetic changes present in the tumor of an individual patient is becoming increasingly important for managing the oncology patient. Our knowledge of the genetic alterations that characterize and predispose to solid tumors continues to expand. Concurrently, the advent of newer technologies such as DNA chips has the potential to enable a more rapid and comprehensive assessment of these changes. The ultimate goal of this new information and technology is to provide sensitive and specific tests that reduce unnecessary procedures and optimize therapy. This review addresses the utility of molecular testing in evaluating cancer. A review of the current technology and hereditary cancer syndromes is also presented.

Prospective multicenter clinical evaluation of AMPLICOR and COBAS AMPLICOR hepatitis C virus tests.

We conducted a multicenter clinical evaluation of the second versions of the manual AMPLICOR and the semiautomated COBAS AMPLICOR tests for hepatitis C virus (HCV) RNA (Roche Molecular Systems, Inc., Pleasanton, Calif.). The performance characteristics of these HCV RNA tests for diagnosis of active viral infection were determined by comparison to anti-HCV serological test results, alanine aminotransferase levels, and liver biopsy histology results. A total of 878 patients with clinical or biochemical evidence of liver disease were enrolled at four hepatology clinics. A total of 1,089 specimens (901 serum and 188 plasma) were tested with the AMPLICOR test. Sensitivity compared to serology was 93.1% for serum and 90.6% for plasma. The specificity was 97% for serum and 93.1% for plasma. A total of 1,084 specimens (896 serum and 188 plasma) were tested with the COBAS test. Sensitivities for serum and plasma were the same as with the AMPLICOR test. The specificity was 97.8% for serum and 96.6% for plasma. Of the 69 specimens with false-positive and false-negative AMPLICOR test results relative to those of serology, alternative primer set (APS) reverse transcription (RT)-PCR analysis showed that the AMPLICOR test provided the correct result relative to the specimens containing HCV RNA in 64 (92.7%) specimens. Similarly, 66 of 67 (98.5%) false-positive and false-negative COBAS test results were determined to be correct by APS RT-PCR analysis. There were no substantive differences in clinical performances between study sites, patient groups, specimen types, storage conditions (-20 to -80 degrees C versus 2 to 8 degrees C), or anticoagulants (EDTA versus acid citrate dextrose) for either test. Both tests showed >99% reproducibility within runs, within sites, and overall. We conclude that these tests can reliably detect the presence of HCV RNA, as evidence of active infection, in patients with clinical or biochemical evidence of liver disease.

Persistence of infectious HIV on follicular dendritic cells.

Follicular dendritic cells (FDCs) trap Ags and retain them in their native state for many months. Shortly after infection, HIV particles are trapped on FDCs and can be observed until the follicular network is destroyed. We sought to determine whether FDCs could maintain trapped virus in an infectious state for long periods of time. Because virus replication would replenish the HIV reservoir and thus falsely prolong recovery of infectious virus, we used a nonpermissive murine model to examine maintenance of HIV infectivity in vivo. We also examined human FDCs in vitro to determine whether they could maintain HIV infectivity. FDC-trapped virus remained infectious in vivo at all time points examined over a 9-mo period. Remarkably, as few as 100 FDCs were sufficient to transmit infection throughout the 9-mo period. Human FDCs maintained HIV infectivity for at least 25 days in vitro, whereas virus without FDCs lost infectivity after only a few days. These data indicate that HIV retained on FDCs can be long lived even in the absence of viral replication and suggest that FDCs stabilize and protect HIV, thus providing a long-term reservoir of infectious virus. These trapped stores of HIV may be replenished with replicating virus that persists even under highly active antiretroviral therapy and would likely be capable of causing infection on cessation of drug therapy.