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1.  Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis 
Background
Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage.
Methods
Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor].
Results
Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate.
Conclusions
The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.
doi:10.1186/1472-6947-11-30
PMCID: PMC3112379  PMID: 21569501
2.  Insulin resistance and glycemic abnormalities are associated with deterioration of left ventricular diastolic function: a cross-sectional study 
Background
Left ventricular diastolic dysfunction (LVDD) is considered a precursor of diabetic cardiomyopathy, while insulin resistance (IR) is a precursor of type 2 diabetes mellitus (T2DM) and independently predicts heart failure (HF). We assessed whether IR and abnormalities of the glucose metabolism are related to LVDD.
Methods
We included 208 patients with normal ejection fraction, 57 (27%) of whom had T2DM before inclusion. In subjects without T2DM, an oral glucose tolerance test (oGTT) was performed. IR was assessed using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). The lower limit of the top quartile of the HOMA-IR distribution (3.217) was chosen as threshold for IR. LVDD was verified according to current guidelines.
Results
IR was diagnosed in 38 (18%) patients without a history of diabetes. The prevalence of LVDD was 92% in subjects with IR vs. 72% in patients without IR (n = 113), respectively (p = 0.013). In the IR group, the early diastolic mitral inflow velocity (E) in relation to the early diastolic tissue Doppler velocity (averaged from the septal and lateral mitral annulus, E'av) ratio (E/E'av) was significantly higher compared to those without IR (9.8 [8.3-11.5] vs. 8.1 [6.6-11.0], p = 0.011). This finding remains significant when patients with IR and concomitant T2DM based on oGTT results were excluded (E/E'av ratio 9.8 [8.2-11.1)] in IR vs. 7.9 [6.5-10.5] in those without both IR and T2DM, p = 0.014). There were significant differences among patients with and without LVDD regarding the HOMA-IR (1.71 [1.04-3.88] vs. 1.09 [0.43-2.2], p = 0.003). The HOMA-IR was independently associated with LVDD on multivariate logistic regression analysis, a 1-unit increase in HOMA-IR value was associated with an odds ratio for prevalent LVDD of 2.1 (95% CI 1.3-3.1, p = 0.001). Furthermore, the E/E'av ratio increases along the glucose metabolism status from normal glucose metabolism (7.6 [6.2-10.1]) to impaired glucose tolerance (8.8 [7.4-11.0]) and T2DM (10.5 [8.1-13.2]), respectively (p < 0.001).
Conclusions
Insulin resistance is independently associated with LVDD in subjects without overt T2DM. Patients with IR and glucose metabolism disorders might represent a target population to prevent the development of HF. Screening programs for glucose metabolism disturbances should address the assessment of diastolic function and probably IR.
doi:10.1186/1475-2840-9-63
PMCID: PMC2964598  PMID: 20950415
3.  Error correction of next-generation sequencing data and reliable estimation of HIV quasispecies 
Nucleic Acids Research  2010;38(21):7400-7409.
Next-generation sequencing technologies can be used to analyse genetically heterogeneous samples at unprecedented detail. The high coverage achievable with these methods enables the detection of many low-frequency variants. However, sequencing errors complicate the analysis of mixed populations and result in inflated estimates of genetic diversity. We developed a probabilistic Bayesian approach to minimize the effect of errors on the detection of minority variants. We applied it to pyrosequencing data obtained from a 1.5-kb-fragment of the HIV-1 gag/pol gene in two control and two clinical samples. The effect of PCR amplification was analysed. Error correction resulted in a two- and five-fold decrease of the pyrosequencing base substitution rate, from 0.05% to 0.03% and from 0.25% to 0.05% in the non-PCR and PCR-amplified samples, respectively. We were able to detect viral clones as rare as 0.1% with perfect sequence reconstruction. Probabilistic haplotype inference outperforms the counting-based calling method in both precision and recall. Genetic diversity observed within and between two clinical samples resulted in various patterns of phenotypic drug resistance and suggests a close epidemiological link. We conclude that pyrosequencing can be used to investigate genetically diverse samples with high accuracy if technical errors are properly treated.
doi:10.1093/nar/gkq655
PMCID: PMC2995073  PMID: 20671025
4.  Reliability of the Bad Sobernheim Stress Questionnaire (BSSQbrace) 
Scoliosis  2007;2(Suppl 1):P1.
doi:10.1186/1748-7161-2-S1-P1
PMCID: PMC3226095  PMID: 17967204
5.  The reliability of the Bad Sobernheim Stress Questionnaire (BSSQbrace) in adolescents with scoliosis during brace treatment 
Scoliosis  2006;1:22.
Background
A new instrument to assess stress scoliosis patients have whilst wearing their brace has been developed. Aim of this study was to test the reliability of this new instrument.
Methods
Eight questions are provided focussing on this topic only, including two questions to test the credibility. A max. score of 24 can be achieved (from 0 for most stress to 24 for least stress). We have proposed a subdivision of the score values as follows: 0–8 (strong stress), 9–16 (medium stress) and 17–24 (little stress).
85 patients were invited to take part in this study and to complete the BSSQbrace questionnaire twice, once at the first presentation and a second after a further three days.
62 patients with an average age of 14,5 years and an average Cobb angle of 40° returned their fully completed questionnaires.
Results
The average stress value was 12,5/24 at the first measurement and 12,4/24 at the second measurement. Ceiling value was 23; floor value 2.
The average stress value was 12,5 / 24 at the first measurement and 12,4 / 24 at the second measurement (from 0 for most stress to 24 for least stress). Ceiling value was 23; floor value 2. There was a correlation of 0,88 (Intraclass Correlation Coefficient) between the values of the two measurements. Cronbach alpha was 0, 97.
Conclusion
The BSSQbrace questionnaire is reliable with good internal consistency and reproducibility. It can be used to measure the coping strategies a patient uses and the impairment a patient feels to have, whilst wearing a brace.
doi:10.1186/1748-7161-1-22
PMCID: PMC1764899  PMID: 17176483
7.  Rapid Typing of Borrelia burgdorferi Sensu Lato Species in Specimens from Patients with Different Manifestations of Lyme Borreliosis 
Journal of Clinical Microbiology  2001;39(3):1130-1133.
To further investigate the pathogenic potential of different Borrelia burgdorferi genospecies, specimens from 27 patients with different manifestations of Lyme borreliosis were analyzed by PCR and reverse line blotting (RLB). In samples from Lyme arthritis patients, B. burgdorferi sensu stricto was predominantly identified, while in patients with neuroborreliosis or acrodermatitis, Borrelia garinii and Borrelia afzelii, respectively, were exclusively detected. The results demonstrate that PCR-RLB is a valuable tool for epidemiological and pathogenetic studies of Lyme borreliosis.
doi:10.1128/JCM.39.3.1130-1133.2001
PMCID: PMC87886  PMID: 11230440

Results 1-7 (7)