Inappropriate Laboratory Testing: Significant Waste Quantified by a Large-Scale Year-Long Study of Medicare and Commercial Payer Reimbursement.

CONTEXT.­: Laboratory testing, beyond what is essential for managing health, is considered low-value care, posing patient risks and wasting resources. Measuring excess testing on a national level is crucial to identify waste and optimize healthcare resource allocation for maximum impact. OBJECTIVE.­: To measure inappropriate laboratory testing and its cost across Medicare and many US commercial payers. DESIGN.­: A retrospective analysis on 2019 claims data measured the frequency of 4 commonly used laboratory tests among 64 million individuals with Medicare and 168 million with commercial insurance. Tests included 25-hydroxy vitamin D, prostate-specific antigen, lipid panel, and hemoglobin A1c. Clinical guidelines, medical literature, and payer recommendations were used to determine appropriate testing frequencies. Costs of excessive testing were calculated using the 2019 clinical lab fee schedule. A targeted analysis of 2022 data confirmed 2019 trends. RESULTS.­: Analysis of ∼84 million tests from ∼1 billion outpatient test claim records revealed that 7% to 51% of tests exceeded recommended frequencies, with some egregious overuse: for example hemoglobin-A1c or prostate-specific antigen every week. The conservative cost estimate for 4 excess tests surpassed $350 million. CONCLUSIONS.­: This extensive study, involving 232 million people, found that 14.4 million of 60.5 million individuals (23.8%) tested had undergone excessive laboratory testing, with likely little benefit and possible harm. Extrapolating findings to all laboratory testing suggests that Medicare alone may have incurred direct excess expenses from $1.95 to $3.28 billion in 2019, without factoring the hidden costs of excessive testing (eg, downstream care). Addressing unnecessary testing is crucial to lowering costs and redirecting resources for greater patient benefit.

Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

Raltegravir is a potent inhibitor of XMRV, a virus implicated in prostate cancer and chronic fatigue syndrome.

BACKGROUND: Xenotropic murine leukemia-related retrovirus (XMRV) is a recently discovered retrovirus that has been linked to human prostate cancer and chronic fatigue syndrome (CFS). Both diseases affect a large fraction of the world population, with prostate cancer affecting one in six men, and CFS affecting an estimated 0.4 to 1% of the population. PRINCIPAL FINDINGS: Forty-five compounds, including twenty-eight drugs approved for use in humans, were evaluated against XMRV replication in vitro. We found that the retroviral integrase inhibitor, raltegravir, was potent and selective against XMRV at submicromolar concentrations, in MCF-7 and LNCaP cells, a breast cancer and prostate cancer cell line, respectively. Another integrase inhibitor, L-000870812, and two nucleoside reverse transcriptase inhibitors, zidovudine (ZDV), and tenofovir disoproxil fumarate (TDF) also inhibited XMRV replication. When combined, these drugs displayed mostly synergistic effects against this virus, suggesting that combination therapy may delay or prevent the selection of resistant viruses. CONCLUSIONS: If XMRV proves to be a causal factor in prostate cancer or CFS, these discoveries may allow for rational design of clinical trials.

Detecting retroviral sequences in chronic fatigue syndrome.

XMRV or xenotropic murine leukemia virus-related retrovirus, a recently discovered retrovirus, has been linked to both prostate cancer and chronic fatigue syndrome (CFS). Recently, the teams of Drs. Shyh-Ching Lo and Harvey Alter discovered the presence of sequences closely related to XMRV in the blood of 86.5% of patients with CFS [1]. These findings are important because since the initial discovery of XMRV in CFS, several studies have failed to find XMRV in specimens collected from CFS patients. While the current study also did not find XMRV in CFS, Lo et al. did detect sequences that belong to polytropic mouse endogenous retroviruses (PMV), which share considerable similarity with XMRV. Criteria for future studies that will help bring greater clarity to the issue of retroviral sequences in CFS are proposed below.

XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors.

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered in human prostate cancers and is the first gammaretrovirus known to infect humans. While gammaretroviruses have well-characterized oncogenic effects in animals, they have not been shown to cause human cancers. We provide experimental evidence that XMRV is indeed a gammaretrovirus with protein composition and particle ultrastructure highly similar to Moloney murine leukemia virus (MoMLV), another gammaretrovirus. We analyzed 334 consecutive prostate resection specimens, using a quantitative PCR assay and immunohistochemistry (IHC) with an anti-XMRV specific antiserum. We found XMRV DNA in 6% and XMRV protein expression in 23% of prostate cancers. XMRV proteins were expressed primarily in malignant epithelial cells, suggesting that retroviral infection may be directly linked to tumorigenesis. XMRV infection was associated with prostate cancer, especially higher-grade cancers. We found XMRV infection to be independent of a common polymorphism in the RNASEL gene, unlike results previously reported. This finding increases the population at risk for XMRV infection from only those homozygous for the RNASEL variant to all individuals. Our observations provide evidence for an association of XMRV with malignant cells and with more aggressive tumors.

Mutational analysis of the N-terminal domain of Moloney murine leukemia virus capsid protein.

Retroviral capsid (CA) proteins contain a structurally conserved N-terminal domain (NTD) consisting of a beta-hairpin and six to seven alpha-helices. To examine the role of this domain in Moloney murine leukemia virus (MoMLV) replication, we analyzed 18 insertional mutations in this region. All mutants were noninfectious. Based on the results of this analysis and our previous studies on additional mutations in this domain, we were able to divide the NTD of MoMLV CA into three functional regions. The first functional region included the region near the N terminus that forms the beta-hairpin and was shown to control normal maturation of virions. The second region included the helix 4/5 loop and was essential for the formation of spherical cores. The third region encompassed most of the NTD except for the above loop. Mutants of this region assembled imperfect cores, as seen by detailed electron microscopy analyses, yet the resulting particles were efficiently released from cells. The mutants were defective at a stage immediately following entry of the core into cells. Despite possessing functional reverse transcriptase machinery, these mutant virions did not initiate reverse transcription in cells. This block could be due to structural defects in the assembling core or failure of an essential host protein to interact with the mutant CA protein, both of which may prevent correct disassembly upon entry of the virus into cells. Future studies are needed to understand the mechanism of these blocks and to target these regions pharmacologically to inhibit retroviral infection at additional stages.

A small loop in the capsid protein of Moloney murine leukemia virus controls assembly of spherical cores.

We report the identification of a novel domain in the Gag protein of Moloney murine leukemia virus (MoLV) that is important for the formation of spherical cores. Analysis of 18 insertional mutations in the N-terminal domain of the capsid protein (CA) identified 3 that were severely defective for viral assembly and release. Transmission electron microscopy of cells producing these mutants showed assembly of Gag proteins in large, flat or dome-shaped patches at the plasma membrane. Spherical cores were not formed, and viral particles were not released. This late assembly/release block was partially rescued by wild-type virus. All three mutations localized to the small loop between alpha-helices 4 and 5 of CA, analogous to the cyclophilin A-binding loop of human immunodeficiency virus type 1 CA. In the X-ray structure of the hexameric form of MLV CA, this loop is located at the periphery of the hexamer. The phenotypes of mutations in this loop suggest that formation of a planar lattice of Gag is unhindered by mutations in the loop. However, the lack of progression of these planar structures to spherical ones suggests that mutations in this loop may prevent formation of pentamers or of stable pentamer-hexamer interactions, which are essential for the formation of a closed, spherical core. This region in CA, focused to a few residues of a small loop, may offer a novel therapeutic target for retroviral diseases.

Functional analysis of hepatitis C virus envelope proteins, using a cell-cell fusion assay.

Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.

T-Cell lymphoblastic lymphoma presenting as bilateral multinodular breast masses: a case report and review of the literature.

Non-Hodgkin lymphoma of T-cell lineage involving the breast is rare. We report on a 41-year-old woman with T-cell lymphoblastic lymphoma who presented with multiple bilateral breast masses. The patient was treated with intensive chemotherapy and mediastinal and whole-brain irradiation. She remains in complete remission 24 months after diagnosis. The clinical, histologic, phenotypic, and cytogenetic features are described, with a review of the literature.

Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting.

Retroviral Gag proteins perform important functions in viral assembly, but are also involved in other steps in the viral life cycle. Conventional mutational analysis has yielded considerable information about domains essential for these functions, yet many regions of gag remain uncharacterized. We used genetic footprinting, a technique that permits the generation and simultaneous analysis of large numbers of mutations, to perform a near-saturation mutagenesis and functional analysis of 639 nucleotides in the gag region of Moloney murine leukemia virus. We report here the resulting functional map defined by eight footprints representing regions of Moloney murine leukemia virus gag, some previously uncharacterized, that are essential for replication. We found that significant portions of matrix and p12 proteins were tolerant of insertions, in contrast to the N-terminal half of capsid, which was not. We analyzed 30 mutants from our library by using conventional methods to validate the footprints. Six of these mutants were characterized in detail, identifying the precise stage at which their replication is blocked. In addition to providing the most comprehensive functional map of a retroviral gag gene, our study demonstrates the abundance of information that can be gleaned by genetic footprinting of viral sequences.

Genetic footprinting of the HIV co-receptor CCR5: delineation of surface expression and viral entry determinants.

Human immunodeficiency virus type 1 (HIV-1) utilizes CD4 as a primary receptor for viral entry and any of several 7-transmembrane chemokine receptors, including CCR5, as a co-receptor. Previous studies have demonstrated that multiple extracellular domains (ECDs) of CCR5 contribute to co-receptor function; here we applied genetic footprinting to CCR5 to confirm and extend those investigations. In genetic footprinting, a duplex oligonucleotide is inserted into the DNA sequence of interest by use of either a bacterial transposase or retroviral integrase. Here, CCR5 mutants were analyzed in bulk for their ability to be expressed on the recipient cell surface and to mediate viral entry of R5 HIV isolates. Most of the approximately 150 CCR5 mutants were not expressed on the cell surface. Of those remaining, 8 were specifically reduced or absent after macrophage (M)-tropic HIV infection, confirming a critical role of ECDs three (extracellular loop 2 or ECL2) and possibly four (ECL3) in viral entry. Mutational and functional analyses of ECD4 (ECL3) suggest it is under severe topological constraint for CCR5 surface expression and are consistent with it contributing to co-receptor function.