Single-level laminoplasty approach to selective dorsal rhizotomy with conus localization by rapid spine MRI.

INTRODUCTION: While selective dorsal rhizotomy (SDR) was originally described as a multilevel approach, single-level approaches are now popularized. Conus localization is beneficial for operative planning in single-level selective dorsal rhizotomy. Our approach to SDR involves minimal exposure for a single-level laminoplasty, preserving one attached interspinous ligament. Pre-operative conus localization is required for this tailored approach to determine the laminoplasty level and dictate rostral or caudal division of the superior spinus ligament. While rapid MRI sequences have been popularized for pediatric cranial imaging, its utility for spinal imagining is less well-described, and specific application for conus localization has not been reported. OBJECTIVE: Illustrate that rapid MRI without sedation is sufficient to identify conus level for tailored single-level laminoplasty SDR. MATERIAL AND METHODS: Patients undergoing SDR from 2014 to 2022 at one institution were reviewed for type of pre-operative MRI (rapid vs full), conus level, procedural time for MRI, and radiology report. The typical rapid MRI has four sequences utilizing single-shot technique (scout, sagittal T2, axial T2, and axial T1) that typically take less than 1 min each of acquisition time, with non-single-shot sequences added periodically in cooperative patients. To include time for patient positioning, pre-scan shimming, procedural incidentals, and other patient-specific variations, MRI procedure length was recorded as documented in the electronic medical record. RESULTS: N = 100 patients had documentation of an MRI for pre-operative imaging. Seventy-nine of these had a rapid MRI, and 21 required a full MRI with anesthesia for their treatment plan. Mean total procedure time for rapid MRI was 21.5 min (median 17). Mean procedure time for MRI under general anesthesia was 91.2 min (median 94). Of patients with rapid MRI imaging, 2/79 had an ambiguous conus level (1 from motion artifact, 1 from spinal hardware) vs 1/21 with a full MRI under anesthesia (due to spinal hardware). CONCLUSION: Rapid spinal MRI without sedation can be used for conus localization in a pediatric population. This may be routinely used as pre-operative imaging for a single-level approach to selective dorsal rhizotomy, without sedation or intubation procedures.

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1.

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses.

Human immunodeficiency virus type 1 isolates with the reverse transcriptase (RT) mutation Q145M retain nucleoside and nonnucleoside RT inhibitor susceptibility.

Q145M, a mutation in a conserved human immunodeficiency virus type 1 reverse transcriptase (RT) region, was reported to decrease susceptibility to multiple RT inhibitors. We report that Q145M and other Q145 mutations do not emerge with RT inhibitors nor decrease RT inhibitor susceptibility. Q145M should not, therefore, be considered an RT inhibitor resistance mutation.

Minority human immunodeficiency virus type 1 variants in antiretroviral-naive persons with reverse transcriptase codon 215 revertant mutations.

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method-ultradeep pyrosequencing (UDPS; 454 Life Sciences)--to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants ("revertant" samples) compared with samples from untreated persons that lack such revertants ("control" samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.

Viral population estimation using pyrosequencing.

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation-maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.

N88D facilitates the co-occurrence of D30N and L90M and the development of multidrug resistance in HIV type 1 protease following nelfinavir treatment failure.

Nelfinavir was once one of the most commonly used protease inhibitors (PIs). To investigate the genetic mechanisms of multidrug resistance in protease isolates with the primary nelfinavir resistance mutation D30N, we analyzed patterns of protease mutations in 582 viruses with D30N from 460 persons undergoing HIV-1 genotypic resistance testing at Stanford University Hospital from 1997 to 2005. Three patterns of mutational associations were identified. First, D30N was positively associated with N88D but negatively associated with N88S. Second, D30N and L90M were negatively associated except in the presence of N88D, which facilitated the co-occurrence of D30N and L90M. Third, D30N+N88D+L90M formed a stable genetic backbone for the accumulation of additional protease inhibitor (PI) resistance mutations. In 16 patients having isolates with more than one combination of mutations at positions 30, 88, and 90, all exhibited one of the steps in the following progression: D30N-->D30N+N88D-->D30N+N88D+L90M-->D30N+N88D+L90M+(L33F+/-I84V or M46I/L+/-I54V). Although nelfinavir is now used less frequently than other PIs, the well-delineated mutational pathway we describe is likely to influence patterns of cross-resistance in viruses from persons who experience virologic failure while receiving this PI.

Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.

Prevalence of darunavir resistance-associated mutations: patterns of occurrence and association with past treatment.

Eleven protease mutations have been associated with reduced susceptibility to darunavir (DRV). We examined the prevalence and covariates of these mutations in 2 populations. Thirty percent of 1175 Northern California patients and 24% of 2744 non-California patients in the Stanford HIV Drug Resistance Database had viruses with 1 or more mutations associated with resistance to DRV. In multivariate analyses, the number of DRV resistance-associated mutations depended on the number of previous protease inhibitors (PIs) administered and on amprenavir/fosamprenavir treatment. Most PI-treated patients should respond favorably to DRV-based salvage therapy.

HIV-1 drug resistance genotype results in patients with plasma samples with HIV-1 RNA levels less than 75 copies/mL.

HIV-1 genotypic resistance test results were obtained on clinical samples from 116 patients with plasma HIV-1 RNA levels of less than 75 copies/mL. Genotype validity was confirmed in 49 of 50 patients with a previous or follow-up genotype. The belief that genotypic resistance testing is unreliable in samples with low-level viremia should be reassessed.

Macrophage inflammatory protein-1 alpha is produced by human multiple myeloma (MM) cells and its expression correlates with bone lesions in patients with MM.

Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a chemokine primarily associated with bone absorption. We examined whether MIP-1alpha was produced by purified fresh tumour cells isolated from bone marrow samples from 31 multiple myeloma (MM) patients. High levels of MIP-1alpha were found in supernatants of myeloma cell cultures. Immunohistochemical staining showed MIP-1alpha in the cytoplasm of myeloma cells. MIP-1alpha mRNA expression was detected in 18 of 31 patients. Bone lesions were seen in 16 of the 18 MIP-1alpha-positive patients but only in six of the 13 MIP-1alpha-negative patients (P = 0.0097,chi2-test). The data indicate that MIP-1alpha is produced by myeloma cells and possibly plays a role in the pathogenesis of bone lesions in MM patients.

A nitric oxide synthase inhibitor, N(G)-nitro-l-arginine-methyl-ester, exerts potent antiangiogenic effects on plasmacytoma in a newly established multiple myeloma severe combined immunodeficient mouse model.

Angiogenesis is one of critical factors in sustaining the growth, invasion and metastasis of certain solid tumours and haematological malignancies such as multiple myeloma (MM). In the present study, we examined the anticancer potential of an inhibitor of nitric oxide synthase (NOS), NG-nitro-l-arginine methyl ester (L-NAME), in a novel severe combined immunodeficient mouse model (KHM mouse) that harbours the highly sanguineous plasmacytoma cell line KHM-4, derived from a patient with highly chemoresistant MM. KHM mice were intraperitoneally administered with either L-NAME, doxorubicin, melphalan, or paclitaxel. A significant reduction in tumour sizes was noted in L-NAME-administered KHM mice while no significant reduction was observed in melphalan- or doxorubicin-administered mice. A profound decrease in angiogenesis was observed in tumour tissues from L-NAME- and paclitaxel-administered KHM mice. A marked decrease in human vascular endothelial cell growth factor (VEGF) levels was identified in tumour tissues from L-NAME-administered KHM mice, strongly suggesting that L-NAME suppressed VEGF production by tumour cells through its inhibition of NOS in tumour cells, resulting in reduced neovasculization in mice leading to the regression of tumour sizes. The present data represent the first observations that certain NOS inhibitors potentially serve as experimental agents for the treatment of chemoresistant MM and plasmacytoma.