Comparative performance evaluation of hepatitis C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of brazilian patients with chronic hepatitis C

<p>Abstract</p> <p>Background</p> <p>Genotyping of hepatitis C virus (HCV) has become an essential tool for prognosis and prediction of treatment duration. The aim of this study was to compare two HCV genotyping methods: reverse hybridization line probe assay (LiPA v.1) and partial sequencing of the NS5B region.</p> <p>Methods</p> <p>Plasma of 171 patients with chronic hepatitis C were screened using both a commercial method (LiPA HCV Versant, Siemens, Tarrytown, NY, USA) and different primers targeting the NS5B region for PCR amplification and sequencing analysis.</p> <p>Results</p> <p>Comparison of the HCV genotyping methods showed no difference in the classification at the genotype level. However, a total of 82/171 samples (47.9%) including misclassification, non-subtypable, discrepant and inconclusive results were not classified by LiPA at the subtype level but could be discriminated by NS5B sequencing. Of these samples, 34 samples of genotype 1a and 6 samples of genotype 1b were classified at the subtype level using sequencing of NS5B.</p> <p>Conclusions</p> <p>Sequence analysis of NS5B for genotyping HCV provides precise genotype and subtype identification and an accurate epidemiological representation of circulating viral strains.</p

Spatial and temporal dynamics of pathogenic Leptospira in surface waters from the urban slum environment

Leptospirosis has emerged as an important urban health problem as slum settlements have expanded worldwide. Yet the dynamics of the environmentally transmitted Leptospira pathogen has not been well characterized in these settings. We used a stratified dense sampling scheme to study the dynamics of Leptospira abundance in surface waters from a Brazilian urban slum community. We collected surface water samples during the dry, intermediate and rainy seasons within a seven-month period and quantified pathogenic Leptospira by quantitative PCR (qPCR). We used logistic and linear mixed models to identify factors that explained variation for the presence and concentration of Leptospira DNA. Among 335 sewage and 250 standing water samples, Leptospira DNA were detected in 36% and 34%, respectively. Among the 236 samples with positive results geometric mean Leptospira concentrations were 152 GEq/mL. The probability of finding Leptospira DNA was higher in sewage samples collected during the rainy season when increased leptospirosis incidence occurred, than during the dry season (47.2% vs 12.5%, respectively, p = 0.0002). There was a marked spatial and temporal heterogeneity in Leptospira DNA distribution, for which type of water, elevation, and time of day that samples were collected, in addition to season, were significant predictors. Together, these findings indicate that Leptospira are ubiquitous in the slum environment and that the water-related risk to which inhabitants are exposed is low. Seasonal increases in Leptospira presence may explain the timing of leptospirosis outbreaks. Effective prevention will need to consider the spatial and temporal dynamics of pathogenic Leptospira in surface waters to reduce the burden of the disease

A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5′UTR — the most highly conserved region of HCV — and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant™ HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant™ HCV Genotype Assay LiPA (74/78, 95%). The concordance between the two methods was 100% for genotype determination (74/74). At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively). Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant™ HCV assay. Genotype “1” subtypes (1a and 1b) were correctly identified by the Versant™ HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping

An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden

Comparison of the efficiency of DNA extraction for three commercial kits from four water types spiked at 1 × 10⁵ <i>Leptospira</i>/mL.

<p>Comparison of the efficiency of DNA extraction for three commercial kits from four water types spiked at 1 × 10⁵ <i>Leptospira</i>/mL.</p

Determination of the optimal volume to be tested by qPCR for different water types: ultrapure water (A), river (B); pond (C) and sewage (D).

<p>Different volumes of each water type were spiked with 1 × 10⁵ leptospires/mL to assess <i>Leptospira</i> recovery. Error bars represent the geometric mean ± SD of the concentrations as determined by qPCR in three independent experiments. Continuous lines connect groups whose average leptospiral DNA concentrations are not statistically significant (p>0.05). Groups whose averages are significantly different (p<0.05) are connected by a dashed line.</p

Determination of the lower limit of detection for water samples obtained ultrapure water (A), river (B), pond (C) and sewage (D).

<p>Ultrapure water (A) was tested with and without the addition of 1 × 10⁷ <i>E</i>. <i>coli</i>/mL. All the samples were spiked with 10⁶ <i>Leptospira</i>/mL and 10-fold serially diluted down to 1 <i>Leptospira</i>/mL. Error bars represent the geometric mean ± SD of three independent experiments.</p