Evaluation of Quantitative Real-Time PCR as a Hepatitis C Virus Supplementary Test After RIBA Discontinuation.

Laboratory testing plays a major role in hepatitis C virus (HCV) diagnosis and patient follow-up. The high false positive rates of HCV screening tests require confirmation through a supplementary test. According to the 2003 CDC guidelines, recombinant immunoblot assay (RIBA) is indispensible to confirm positive screening results and differentiate biologic false positivity from true HCV exposure. However, RIBA has been permanently discontinued since 2011. In the 2013 update of its guidelines, CDC called for further studies on HCV laboratory testing without RIBA. In this study, we analyzed the applicability of quantitative real-time PCR (qPCR) as a supplementary HCV diagnostic test. By comparing our HCV testing performances before and after RIBA discontinuation, we found that omitting RIBA has no significant effect on the accurate and efficient identification of HCV infection, provided that HCV antibody signal-to-cutoff ratio is considered. Furthermore, we proposed a new HCV testing algorithm that incorporates semiquantitative assessment of HCV antibody positivity and HCV viral load measurement by qPCR. By following the algorithm, we were able to address confirmation of positive HCV screening results and to provide useful information generally required by clinicians, including the needs of further laboratory testing or clinical follow-up, as well as HCV viral titers.

Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2α.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

Sensitive deep-sequencing-based HIV-1 genotyping assay to simultaneously determine susceptibility to protease, reverse transcriptase, integrase, and maturation inhibitors, as well as HIV-1 coreceptor tropism.

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3' end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals.

Spectrum of neurological and survival outcomes in pyruvate dehydrogenase complex (PDC) deficiency: lack of correlation with genotype.

Pyruvate dehydrogenase complex (PDC) deficiency is a relatively common mitochondrial disorder that primarily presents with neurological manifestations and lactic acidemia. We analyzed the clinical outcomes and neurological features of 59 consented symptomatic subjects (27 M, 32 F), who were confirmed to have PDC deficiency with defined mutations in one of the genes of PDC (PDHA1, n = 53; PDHB, n = 4; DLAT, n = 2), including 47 different mutations, of which 22 were novel, and for whom clinical records and/or structured interviews were obtained. 39% of these subjects (23/59) have died. Of these, 91% (21/23) died before age 4 years, 61% (14/23) before 1 year, and 43% (10/23) before 3 months. 56% of males died compared with 25% of females. Causes of death included severe lactic acidosis, respiratory failure, and infection. In subjects surviving past 6 months, a broad range of intellectual outcomes was observed. Of 42 subjects whose intellectual abilities were professionally evaluated, 19% had normal or borderline intellectual ability (CQ/IQ ≥ 70), 10% had mild intellectual disability (ID) (CQ/IQ 55-69), 17% had moderate ID (CQ/IQ 40-54), 24% had severe ID (CQ/IQ 25-39) and 33% had profound ID (CQ/IQ<25). Assessment by parents was comparable. Of 10 subjects who reached age 12 years, 9 had had professional IQ assessments, and only 4 had IQs ≥ 70 (only 2 of these 4 had assessments after age 12 years). The average outcome for females was severe-to-profound ID, whereas that of males was mild-to-moderate ID. Of subjects for whom specific neurological data were available, the majority had hypotonia (89%), and hypertonia or mixed hyper-/hypotonia (49%) were common. Seizures (57%), microcephaly (49%), and structural brain abnormalities including ventriculomegaly (67%) and agenesis, dysgenesis, or hypoplasia of the corpus callosum (55%) were common. Leigh syndrome was found in only 35%. Structural brain abnormalities were more common in females, and Leigh syndrome was more common in males. In a subgroup of 16 ambulatory subjects >3.5 years in whom balance was evaluated, ataxia was found in 13. Peripheral neuropathy was documented in 2 cases but not objectively evaluated in most subjects. Outcomes of this population with genetically confirmed PDC deficiency are heterogeneous and not distinctive. Correlations between specific genotypes and outcomes were not established. Although more females survive, related to the prevalence of X-linked PDHA1 mutations, symptomatic surviving females are generally more severely impaired cognitively and have a different pattern of neurological impairment compared to males. Neonatal or infant onset of symptoms was associated with poor outcomes. Males with PDHA1 mutations and low fibroblast PDC activity were less likely to survive beyond infancy. Recurrence rate in siblings of subjects with PDHA1 mutation was less than 5%. Paradoxically, in this retrospective review, potential factors considered possibly relevant to development, such as in vitro PDC activity, specific mutations, use of ketogenic diets, supplements, or medications, were generally not confirmed to be significantly correlated with objective outcomes of survival or neuro-cognitive function. Therefore, the basis of variability of these outcomes remains largely undetermined.

Procedure-specific preoperative red blood cell preparation and utilization management in pediatric surgical patients.

BACKGROUND: Data-driven practices in preoperative red blood cell (RBC) preparation for pediatric surgical procedures are not well established. Adaptation of established adult preparation guidance methods to pediatric populations may improve perioperative RBC utilization. STUDY DESIGN AND METHODS: A retrospective audit of preoperative RBC preparation volumes (Vp) and intraoperative RBC transfusion volumes (Vt) for pediatric surgical procedures was undertaken at a large children's hospital from January to June 2006. RBC preparation-to-transfusion volume (mL/kg) ratios (P:T) were calculated for all surgeries, subspecialties, and select procedures. P:T equals Vp divided by Vt. Resulting P:Ts were compared to a target P:T of 2:1. A model for maximum procedure-specific Vp (Vp-max) defined Vp-max as the RBC transfusion volume able to meet the needs of 80% of patients undergoing an individual surgical procedure. Vp-max values were applied to the study data set to predict the impact on P:Ts and Vp. RESULTS: RBCs were prepared for 332 surgical procedures and transfused during 113 procedures. P:T was 3.5:1 for total surgical procedures (subspecialty range, 2.7:1-46:0), exceeding the 2:1 target. Vp-max modeling for spinal fusion, craniotomy for neoplasia, craniotomy for seizure, and craniosynostectomy yielded P:T ratios of 1.5:1, 1.5:1, 1.7:1, and 1.0:1, respectively, predicting a 30% decrease in Vp for these four surgical procedures. CONCLUSIONS: P:Ts for pediatric surgical procedures at this institution indicate potentially excessive preoperative RBC preparations. Determination of data-driven procedure-specific Vp may increase the efficiency of preoperative RBC preparation practices.

Von Hippel-Lindau (VHL) disease: an update on the clinico-pathologic and genetic aspects.

von Hippel-Lindau (VHL) disease is an inherited multisystem familial cancer syndrome caused by mutations of the VHL gene on chromosome 3p25. A wide variety of neoplastic processes are known to be associated with VHL disease. The consequences of the VHL mutations and the pathway for tumor development continue to be elucidated. This paper will detail the variety of tumors associated with VHL disease and discuss the genetic mechanisms that lead to the predisposition for neoplasia.

Interference of Therapeutic Monoclonal Antibodies With Routine Serum Protein Electrophoresis and Immunofixation in Patients With Myeloma: Frequency and Duration of Detection of Daratumumab and Elotuzumab.

OBJECTIVES: We investigated the frequency and pattern of detection of therapeutic monoclonal antibodies (t-mAbs) daratumumab and elotuzumab by routine serum protein electrophoresis (SPE) and immunofixation (IF) in treated patients with myeloma. METHODS: Detection of t-mAb was assessed in 22 patients by retrospective review of SPE/IF ordered prior to, during, and after 26 individual courses of therapy. RESULTS: t-mAb was distinguishable from M-protein in 16 of 26 courses, with daratumumab detected in nine of nine and elotuzumab in six of seven patients. t-mAb was detected on first follow-up SPE/IF in 12 patients, with earliest detection 7 days after therapy initiation and latest detection 70 days after therapy. t-mAb persisted throughout induction therapy in most patients, with loss of detection during maintenance daratumumab. CONCLUSIONS: When distinguishable from M-protein, t-mAbs are detectable in 93% of treated patients as soon as 7 days after the initial dose and are consistently observed throughout induction therapy, warranting increased monitoring and careful interpretation of SPE/IF.

Validation of hemolysis index thresholds optimizes detection of clinically significant hemolysis.

OBJECTIVES: Automated hemolysis index (HI) measurement has standardized the identification and gradation of sample hemolysis. METHODS: This study evaluates whether clinically significant changes in the concentration of intracellular analytes occur at manufacturer-recommended automated HI thresholds (HI ≥3, >25 mg/dL hemoglobin). RESULTS: Adult outpatient results for serum potassium (K+), magnesium (Mg), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) were analyzed. Mean ± SD analyte concentration and distribution within the reference interval (RI) were calculated for each HI group (1-7). Potassium results with an HI of 4 or more demonstrated clinically significant differences (≥0.5 mmol/L) in mean K+ concentration and RI classification compared with non-hemolyzed samples (HI = 1). LDH and AST showed clinically significant differences (+20%) for an HI of 3 or more. For Mg, only the group with an HI of 7 demonstrated a clinically significant difference (>25%); however, the number was low. CONCLUSIONS: Mean measured potassium concentrations are not clinically significantly affected by hemolysis at the manufacturer-recommended HI threshold, while AST and LDH are. Aligning reporting of sample hemolysis with clinically significant changes provides clinically meaningful alerts regarding this common pre-analytic error.

Next-Generation Sequencing to Help Monitor Patients Infected with HIV: Ready for Clinical Use?

Given the extreme variability of the human immunodeficiency virus (HIV) and its ability to replicate as complex viral populations, HIV variants with reduced susceptibility to antiretroviral drugs or with specific coreceptor tropism (CCR5 and/or CXCR4) may be present as minority members of the viral quasispecies. The sensitivity of current HIV genotypic or phenotypic assays is limited, and thus, these tests usually fail to detect low-abundance viral variants. Next-generation (deep) sequencing (NGS) produces an enormous amount of information that allows the detection of minority HIV variants at levels unimaginable using standard Sanger sequencing. NGS technologies continue to evolve, opening new and more affordable opportunities to implement this methodology in clinical laboratories, and HIV is not an exception. The ample use of a battery of more effective antiretroviral drugs, together with careful patient monitoring based on HIV resistance testing, has resulted in HIV-infected patients whose disease is usually well-controlled. The vast majority of adherent patients without detectable resistance become virologically suppressed; however, a subset of these patients with undetectable resistance by standard methods may fail antiretroviral therapy, perhaps due to the presence of minority HIV-resistant variants. Novel NGS-based HIV assays with increased sensitivity for identifying low-level drug resistance and/or coreceptor tropism may play an important role in the success of antiretroviral treatments.

Comparison of diabetes mellitus and insulin resistance screening methods for women with polycystic ovary syndrome.

OBJECTIVE: To compare screening strategies for type 2 diabetes mellitus (DM), impaired glucose tolerance (pre-DM), and insulin resistance (IR) in women with polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: Academic reproductive endocrinology practice. PATIENT(S): Adult women with PCOS (n = 111). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Subjects were screened for pre-DM and DM using a 2-hour glucose tolerance test (GTT), hemoglobin A1c (HbA1c), or fasting plasma glucose (FPG) and for IR using homeostasis model assessment (HOMA), insulin levels (fasting and 2 hours after 75 glucose load), or obesity. Screening approaches were compared using positive and negative percent agreement and Cohen's kappa (κ). RESULT(S): DM and pre-DM were diagnosed by GTT in 4% and 20% of subjects, respectively. Screening with FPG failed to identify 41% of pre-DM and 20% of DM subjects. GTT and HbA1c had only fair agreement (κ = 0.29). IR was diagnosed in 24% of subjects with pre-DM or DM and in 56% of the remaining subjects using HOMA and insulin levels. HOMA and elevated insulin levels demonstrated substantial agreement for detecting IR (κ = 0.70-0.73). Obesity demonstrated fair to slight agreement (κ = 0.33-0.18). CONCLUSION(S): Women with PCOS should be screened for Pre-DM and DM using GTT or HbA1c, and those with Pre-DM or DM are presumed to have IR. In the rest, IR can be detected using either HOMA or insulin levels.

Two cases of agnathia (otocephaly): with review of the role of fibroblast growth factor (FGF8) and bone morphogenetic protein (BMP4) in patterning of the first branchial arch.

Agnathia (otocephaly) is a sporadic malformation characterized by agenesis of the mandible with characteristic dysmorphologic sequelae. We compare the prenatal presentations and dysmorphologic abnormalities of 2 female fetuses with agnathia. Fetus 1 was delivered at 33 weeks' gestational age and showed agnathia with characteristic sequelae of microstomia; microglossia; persistent buccopharyngeal membrane; and ventrally placed, malformed external ears. Fetus 2 was delivered at 32 weeks' gestational age and exhibited agnathia, astomia, and microglossia; in contrast to fetus 1, however, the external ears were laterally placed, low set, and malformed. For both fetuses, tridimensional computed tomographic scan showed the unique complete absence of the mandible. Additional malformations were documented and differed between the fetuses. We discuss the current molecular mechanisms implicated in 1st branchial arch patterning, particularly the impact of bone morphogenetic protein and fibroblast growth factor 8, and how these findings have the potential to explain the spectrum of abnormalities present in these 2 fetuses with agnathia without associated holoprosencephaly.