Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

Evaluation of the new cobas® HCV genotyping test based on real-time PCRs of three different HCV genome regions.

BACKGROUND: Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to evaluate the performance of the recently introduced cobas® HCV GT assay (Roche) and to compare it to two comparator assays. METHODS: The cobas® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE® HCV 5'NC Genotyping Kit (Siemens) and the VERSANT® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included. RESULTS: When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample. CONCLUSIONS: The cobas® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.

Conditions of High Resolution Melting Analysis on the Cobas z480 Instrument for the Genotyping of VKORC1 in the Clinical Routine Laboratory.

BACKGROUND: High resolution melting (HRM) of amplicons is a simple method for genotyping of single nucleotide polymorphisms (SNPs). Albeit many applications reported, HRM seems to be rarely used in clinical laboratories. The suitability of HRM-PCR for the clinical laboratory was investigated for genotyping of SNPs of the vitamin K epoxide reductase complex unit 1 gene. METHODS: About 100 DNA samples were analyzed by two different HRM-PCRs on the Cobas z480 instrument and compared with a PCR with fluorescently labeled probes (HybProbe-PCR) on the LightCycler 2.0 instrument as reference. RESULTS: Reliable genotyping with 100% matching results was obtained, when the amplicon size was small (63 bp) and DNA input was limited by e.g., sample dilution with salt-free water. CONCLUSIONS: DNA extracted by differing methods may be used for genotyping by HRM-PCR. Compared with HybProbe-PCR, HRM-PCR on the Cobas z480 instrument allows for higher through-put, however, at the cost of a higher degree of laboratory standardization and a slower turnaround.

Reliable analysis of the single nucleotide polymorphism of lactase persistence LPH(-13910) C/T from saliva derived DNA: validation of a standardized saliva collection system.

BACKGROUND: The purpose of this study was to investigate the applicability of the Greiner Saliva Collection System (SCS) to obtain human genomic DNA for the analysis of single nucleotide polymorphisms (SNP) in the clinical routine laboratory. METHODS: Saliva and EDTA-blood were collected pair-wise from 112 participants. DNA was prepared by two automated procedures (MagNA Pure LC or MagNa Pure compact) and analyzed by UV-spectrophotometry and real-time PCR. RESULTS: Mean saliva derived DNA concentration was 52.7 ng/µL ± 36.4 (1000 µL, MagNA Pure LC) and 9.2 ng/µL ± 5.6 (200 µL, MagNA Pure compact) with A260/A280 ratios of 1.9 ± 0.1 and 2.1 ± 0.3 for MagNA Pure LC and MagNA Pure compact, respectively. SNP analysis for caucasian adult type lactase persistence showed a 100% success rate from saliva derived DNA and as reference from blood derived DNA. Matching genotypes were obtained in each sample pair. CONCLUSIONS: Saliva obtained with the standardized SCS yielded sufficient amounts of DNA in high purity and was found to represent a suitable and reliable source of human DNA for SNP analysis in the clinical routine laboratory.

A case of acute myeloid leukemia (AML) with an unreported combination of chromosomal abnormalities: gain of isochromosome 5p, tetrasomy 8 and unbalanced translocation der(19)t(17;19)(q23;p13).

BACKGROUND: Acute myeloid leukemia (AML) comprises a spectrum of myeloid malignancies which are often associated with distinct chromosomal abnormalities, and the analysis of such abnormalities provides us with important information for disease classification, treatment selection and prognosis. Some chromosomal abnormalities albeit recurrent are rare such as tetrasomy 8 or isochromosome 5p. In addition, erratic chromosomal rearrangements may occur in AML, sometimes unbalanced and also accompanied by other abnormalities. Knowledge on the contribution of rare abnormalities to AML disease, progression and prognosis is limited.Here we report a unique case of acute monoblastic leukemia with gain of i(5)(p10), tetrasomy 8, an unbalanced translocation der(19)t(17;19)(q23;p13.3) and mutated NPM1. RESULTS: Bone marrow cells were examined by conventional karyotyping, fluorescence in situ hybridization (FISH) and mutation analysis at diagnosis and follow-up. At diagnosis we detected trisomy 8, an unbalanced translocation der(19)t(17;19)(q23;p13.3) and mutated NPM1. During the course of the disease we observed clonal evolution with gain of i(5)(p10), tetrasomy 8 and eventually duplication of der(19)t(17;19)(q23;p13.3). By using the der(19)t(17;19) as clonal marker, we found that i(5)(p10) and tetrasomy 8 were secondary genetic events and that tetrasomy 8 had clonally evolved from trisomy 8. CONCLUSIONS: This case of acute monoblastic leukemia presents a combination of rare chromosomal abnormalities including the unbalanced translocation der(19)t(17;19)(q23;p13.3), hitherto un-reported in AML. In addition, our case supports the hypothesis of a step-wise clonal evolution from trisomy 8 to tetrasomy 8 in AML. Reporting and collecting data of rare chromosomal abnormalities will add information to AML disease, progression and prognosis, and may eventually translate to improved patient management.

Genotypic prediction of human immunodeficiency virus type 1 tropism by use of plasma and peripheral blood mononuclear cells in the routine clinical laboratory.

We developed a sequencing assay for genotypic HIV-1 tropism determination. The assay allows examination of HIV RNA from plasma and HIV DNA from peripheral blood mononuclear cells (PBMC), including PBMC samples from patients with undetectable viral loads. Assessment of 100 pairs of plasma and PBMC samples showed a high concordance of 90%. With the limitations of population-based sequencing, the assay was found to be robust and suitable for the routine clinical laboratory.

Alkaline phosphatase predicts relapse in chronic hepatitis C patients with end-of-treatment response.

AIM: To investigate relapse predictors in chronic hepatitis C (CHC) patients with end-of-treatment response (ETR), after pegylated interferon-alpha (PegIFN-alpha) and ribavirin treatment. METHODS: In a retrospective study we evaluated a spectrum of predictors of relapse after PegIFN-alpha and ribavirin treatment in 86 CHC patients with ETR. Viral loads were determined with real-time reverse transcription polymerase chain reaction. Hepatitis C virus genotyping was performed by sequencing analysis. Patients with genotype 1 were treated for 48 wk with 180 microg PegIFN-alpha2a or 1.5 microg/kg PegIFN-alpha2b once weekly plus ribavirin at a dosage of 1000 mg/d for those under 75 kg or 1200 mg/d for those over 75 kg. Patients with genotypes 2 and 3 were treated for 24 wk with 180 microg PegIFN-alpha2a or 1.5 microg/kg PegIFN-alpha2b once weekly plus ribavirin at a dosage of 800 mg/d. RESULTS: In all ETR patients, binary logistic regression analysis identified absence of complete early virological response (cEVR) (OR 27.07, 95% CI: 3.09-237.26, P < 0.005), serum alkaline phosphatase (ALP) levels prior to therapy < 75 U/L (OR: 6.16, 95% CI: 2.1-18.03, P < 0.001) and body mass index > 26 kg/m(2) (OR: 8.27, 95% CI: 2.22-30.84, P < 0.005) as independent predictors of relapse. When cEVR patients were analyzed exclusively, ALP prior to therapy < 75 U/L remained the only predictor of relapse. CONCLUSION: Lower levels of ALP prior to, during and after therapy seem to be associated with a higher risk of relapse in CHC patients with ETR.

Serum levels of N-terminal pro-B-type natriuretic peptide are associated with allograft function in recipients of renal transplants.

BACKGROUND: B-type natriuretic peptide (BNP) plays a key role in the regulation of volume homeostasis, and elevated blood levels of BNP are associated with end-stage renal disease. Renal transplantation leads to a decrease of elevated BNP levels with established graft function. Assessment of N-terminal pro-BNP (NT-proBNP) is established as reflecting volume homeostasis, and we therefore studied the relationship between NT-proBNP and allograft function in a prospective study. METHODS: NT-proBNP was assessed in 76 patients with end-stage renal disease undergoing renal transplantation. Patients were grouped according to immediate or delayed graft function. The degree of allograft function was assessed from the estimated glomerular filtration rate according to the MDRD formula. RESULTS: In patients with immediate graft function (n = 48), median NT-proBNP decreased immediately after transplantation; in patients with delayed function (n = 28), median NT-proBNP first increased and then decreased as function improved. Patients with early acute rejection showed significantly higher NT-proBNP levels prior to transplantation than patients without rejection. NT-proBNP levels measured 2 or 3 weeks post-transplant were significantly correlated with the estimated glomerular filtration rate 1 year after transplantation. CONCLUSIONS: An association was observed between renal allograft function and post-transplant levels of NT-proBNP. The association was not found to be a useful general predictor for graft function in individual patients in a clinical setting, as the range of NT-proBNP levels measured was too wide.

Lymphocyte imbalance in vitiligo patients indicated by elevated CD4+/CD8+ T-cell ratio.

Vitiligo is a disorder of pigmentation associated with an autoimmune-mediated loss of melanocytes from the epidermis. Humoral immunity and the involvement of cellular immunity have been investigated in the pathogenesis of vitiligo. We evaluated the role of pro-inflammatory cytokines and lymphocyte fractions in peripheral blood in a cohort of Austrian patients with vitiligo. Morning blood samples from 40 patients with vitiligo were collected. Twenty-one patients had active and 19 had stable vitiligo disease. All patients were suffering from non-segmental vitiligo at different stages of the disease. Sixteen persons presented with an additional autoimmune thyroid disease. To evaluate a possible involvement of proinflammatory cytokines in vitiligo we measured sTNF-RI (soluble tumour necrosis factor receptor I), IL-6 and additionally CIC (circulating immune complexes). We compared these findings to the data from matched normal persons. To investigate the mechanisms of cellular immunity, peripheral blood cell count and lymphocyte subtype analysis by flow cytometry were done. sTNF-RI, IL-6 and CIC serum levels were in the normal range. In the patient group median sTNF-RI level was 1.5 ng/ml and median CIC level was 35.2 microg/ml, and no statistically significant differences to the control group were observed. Median IL-6 level in vitiligo patients was 2.7 pg/ml and in the normal range-but higher than the median level of 0.5 pg/ml observed in normal persons (p < 0.001). Absolute and relative counts of lymphocyte subtypes were normal. The ratio of CD4+/CD8+ T-cells had an elevated median value of 2.6 [quartiles 2.0; 3.1]. 61% of the vitiligo patients had a ratio higher than 2.4, which was the normal cut-off point. In most vitiligo patients the balance of cytotoxic/suppressor and helper/inducer T-cells in peripheral blood is disturbed which might lead to a predominance of T-cell subtypes in the intracutaneous site of autoimmune melanocyte loss.

Genotypic antiretroviral resistance testing for human immunodeficiency virus type 1 integrase inhibitors by use of the TruGene sequencing system.

A sequencing assay for detection of mutations conferring resistance to human immunodeficiency virus type 1 (HIV-1) integrase inhibitors raltegravir and elvitegravir was developed using the automated TruGene sequencing system. The assay returned clear sequencing results for samples with >or=500 RNA copies/ml for mutation detection and HIV-1 subtyping across a spectrum of HIV-1 subtypes.

Hyperthyroidism affects arterial stiffness, plasma NT-pro-B-type natriuretic peptide levels, and subendocardial perfusion in patients with Graves' disease.

BACKGROUND: Arterial stiffness is thought to play a critical role in the pathogenesis of cardiovascular events, and in hyperthyroidism increased cardiovascular event rates have been reported. AIM: To investigate markers of systemic arterial stiffness, volume homeostasis, and subendocardial perfusion and its interrelationship in patients with Graves' disease (GD) in hyperthyroidism and euthyroidism. METHOD: Aortic augmentation index (AIx@75) as a measure of systemic arterial stiffness and subendocardial viability ratio (SEVR) as a surrogate measure of subendocardial perfusion were assessed by applanation tonometry in 59 patients with GD in hyperthyroidism and euthyroidism, and measurements were compared to plasma levels of NT-pro-B-type natriuretic peptide (NT-ProBNP). RESULTS: AIx@75 and NT-ProBNP levels were significantly increased in hyperthyroidism compared to euthyroidism and were positively correlated with each other. SEVR was significantly decreased in hyperthyroidism compared to euthyroidism, mainly due to increased heart rates as shown by the heart rate-corrected SEVR75. CONCLUSIONS: In hyperthyroidism, patients with GD exhibited increased systemic arterial stiffness, paralleled by increased levels of NT-ProBNP, a marker of volume overload. The decreased subendocardial perfusion in hyperthyroidism seemed to be mainly due to increased heart rates. The observed unfavorable hemodynamic alterations in hyperthyroidism may serve to explain increased cardiovascular event rates in patients with GD.

Prevalence, severity and predictors of HOMA-estimated insulin resistance in diabetic and nondiabetic patients with end-stage renal disease.

BACKGROUND: Prevalence of insulin resistance (IR) is increased in type 2 diabetes and in end-stage renal disease (ESRD). IR is associated with advanced atherosclerosis and is an independent predictor for cardiovascular disease in diabetes and ESRD patients. We investigated prevalence, severity, predictors and relation to vascular diseases by the homeostasis model assessment (HOMA-IR) in diabetic and nondiabetic ESRD patients. METHODS: ESRD patients with type 2 diabetes (n = 27) and nondiabetic ESRD patients (n = 35) were included in the study. IR was assessed with the HOMA-IR using fasting glucose and insulin levels. Additionally, serum levels of C-peptide, HbA1c, triglycerides, cholesterol and C-reactive protein and blood pressure were assessed. RESULTS: Median HOMA-IR was significantly higher in the diabetic ESRD patients than in the nondiabetic ESRD patients (6.3 [range 0.7-61.7] vs. 2.4 [range 0.3-5.7]; p < 0.001). Systolic blood pressure and triglycerides were significantly higher in patients with higher HOMA-IR, whereas HDL cholesterol was significantly lower in those patients. Only nondiabetic patients with increased HOMA-IR had significantly higher C-peptide levels than those with lower HOMA-IR (14.9 + 5.7 vs. 9.0 + 4.3, p = 0.004). Vascular disease prevalence was significantly higher in diabetic patients with higher HOMA-IR than in those with lower HOMA-IR. CONCLUSIONS: Prevalence and severity of HOMA-IR was greater in diabetic ESRD patients than in those without diabetes. In diabetic patients low HDL cholesterol was the only predictor for higher HOMA-IR, whereas in nondiabetic patients a high C-peptide level was the only predictor for higher HOMA-IR. The prevalence of vascular diseases is associated with higher HOMA-IR in ESRD patients.

Genotyping of the lactase-phlorizin hydrolase -13910 polymorphism by LightCycler PCR and implications for the diagnosis of lactose intolerance.

BACKGROUND: Hypolactasia and lactose intolerance are common conditions worldwide. Hypolactasia seems to be strongly correlated with genotype C/C of the genetic variant C-->T(-13910) upstream of the lactase phlorizin hydrolase (LPH) gene. We developed a rapid genotyping assay for LPH C-->T(-13910) and investigated the relationship of positive lactose breath hydrogen test (LBHT) results suggesting lactose intolerance with LPH C-->T(-13910) genotype. METHODS: Using automated DNA purification on the MagNA Pure LC and real-time PCR on the LightCycler, we examined samples from 220 individuals to estimate genotype frequencies; we then determined LPH C-->T(-13910) genotype in samples from 54 Caucasian patients with a positive LBHT result and symptoms of lactose intolerance. RESULTS: Genotyping of 220 individuals revealed frequencies of 21.4%, 41.8%, and 36.8% for genotypes C/C, C/T, and T/T. Of the patients with positive LBHT results, only 50% had the C/C genotype suggestive of primary adult hypolactasia in our study population. The other patients had various degrees of secondary hypolactasia or symptoms of lactose intolerance. Patients with C/C genotype had a mean (SD) peak H2 increase in the LBHT [108 (58) ppm] that was significantly higher than in patients with the C/T [65 (54) ppm] and T/T [44 (34) ppm] genotypes. CONCLUSIONS: The new real-time PCR assay provides a rapid, labor-saving means for the genotyping of LPH C-->T(-13910). Use of the assay may assist in differentiating patients with primary hypolactasia from those with secondary hypolactasia and lactose intolerance, who may need further clinical examinations to diagnose their underlying primary diseases.

Rapid diagnosis of adenoviral keratoconjunctivitis by a fully automated molecular assay.

OBJECTIVE: To establish and evaluate a new test system for rapid detection and diagnosis of adenoviral keratoconjunctivitis. DESIGN: After establishment of the molecular assay, 52 conjunctival smears were studied. PARTICIPANTS: Samples were derived from patients with a clinical presentation compatible with keratoconjunctivitis. METHODS: A molecular assay for detection of human adenovirus (HAdV) based on automated nucleic acid extraction and real time polymerase chain reaction was established and evaluated. The new assay included a heterologous internal control. MAIN OUTCOME MEASURES: Statement about the presence or absence of adenoviral DNA in the specimen. RESULTS: The amplification efficiency was found to be 100%. The detection limit was calculated to be 116 copies per LightCycler capillary. When clinical specimens were tested, 15 of 52 conjunctival smears were found to be positive for HAdV DNA. The internal control was detected in all samples. CONCLUSIONS: The new molecular assay proved to be suitable for rapid diagnosis of adenoviral keratoconjunctivitis in the routine diagnostic laboratory.

Diurnal variation of arterial stiffness and subendocardial perfusion noninvasively assessed using applanation tonometry in healthy young men.

BACKGROUND: Arterial stiffness is at least partially controlled by vascular tone. Vascular tone and underlying physiological processes, e.g. sympathetic activity, have been shown to follow diurnal variations. METHODS: This study investigated whether arterial stiffness and perfusion of subendocardial myocardium relative to cardiac workload show diurnal variations under physiological conditions. The aortic augmentation index (AIx) and subendocardial viability ratio (SEVR) were measured noninvasively in 26 healthy young men (27.6 +/- 3.4 years) using applanation tonometry at three different times (8:00, 12:00, 17:00) during one day. RESULTS: Mean AIx was significantly higher and mean SEVR significantly lower at 8:00 than at the later times. No significant differences were found between mean AIx and mean SEVR at 12:00 and at 17:00. CONCLUSIONS: The observed diurnal variations of AIx and SEVR will be of value when applanation tonometry is used in human research. In order to arrive at comparable data in longitudinal investigations, measurements should be made at similar times during the course of a day. In addition, our observation should assist in studies in which novel pharmacological compounds with activity on the vasculature are investigated.

Hypoplastic thyroid, growth hormone deficiency, corneal opacities, cataract and hyperkeratotic skin disease: a possible new ichthyosis syndrome associated with endocrinopathies.

A 56 year old man presented with ichthyosis vulgaris since early childhood, clinically characterised by fine scaling of the trunk and hyperkeratotic scales on the exterior surfaces of the upper and lower extremities. The patient also showed hypothyroidism due to hypoplastic thyroid, cataract, hypercholesterinemia with concommitant arcus cornealis and biliary concrements. Renal lithiasis caused by calcio-oxalate was additionally present. Endocrinological screening revealed growth hormone deficiency in the 1.55 m tall man-(secondary) osteoporosis was observed. The clinical symptomatology indicates that this case cannot be considered as a subtype of the inherited ichthyosis group, but suggests a new syndrome as a separate nosologic entity.