Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target (‘tester’). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens (‘driver’). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies

Next-generation sequencing reveals large connected networks of intra-host HCV variants

Background: Next-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host. Results: Intra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient. The distance between any two variants calculated over the component correlated strongly with nucleotide distances (r = 0.9499; p = 0.0001), a better correlation than the one obtained with Neighbour-Joining trees (r = 0.7624; p = 0.0001). In each patient, components were well separated, with the average distance between (6.53%) being 10 times greater than within each component (0.68%). The ratio of nonsynonymous to synonymous changes was calculated and some patients (6.9%) showed a mixture of networks under strong negative and positive selection. All components were robust to in silico stochastic sampling; even after randomly removing 85% of all reads, the largest connected component in the new subsample still involved 82.4% of remaining nodes. In vitro sampling showed that 93.02% of components present in the original sample were also found in experimental replicas, with 81.6% of reads found in both. When syringe-sharing transmission events were simulated, 91.2% of all simulated transmission events seeded all components present in the source. Conclusions: Most intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance

Drug-resistance of a viral population and its individual intra-host variants during the first 48 hours of therapy

Using HCV and IFN-resistance as a proof of concept, we have devised a new methodology for calculating the effect of a drug over a viral population and the resistance of its individual intra-host variants. By means of next-generation sequencing, HCV variants were obtained from sera collected at 9 time-points from 16 patients during the first 48 hours after injection of IFN-α. IFN-resistance coefficients were calculated for individual variants using changes in their relative frequencies, and for the entire intra-host viral population using changes in viral titer during the initial 48 hours. Population-wide resistance and presence of IFN-resistant variants were highly associated with pegIFN-α2a/RBV treatment outcome at week 12 (p = 3.78×10-5 and 0.0114, respectively). This new method allows an accurate measurement of resistance based solely on changes in viral titer or the relative frequency of intra-host viral variants during a short observation time

Acute Hepatitis B Outbreaks in 2 Skilled Nursing Facilities and Possible Sources of Transmission: North Carolina, 2009–2010

Objective. Acute hepatitis B virus (HBV) infections have been reported in long-term care facilities (LTCFs), primarily associated with infection control breaks during assisted blood glucose monitoring. We investigated HBV outbreaks that occurred in separate skilled nursing facilities (SNFs) to determine factors associated with transmission. Design. Outbreak investigation with case-control studies. Setting. Two SNFs (facilities A and B) in Durham, North Carolina, during 2009–2010. Patients. Residents with acute HBV infection and controls randomly selected from HBV-susceptible residents during the outbreak period. Methods. After initial cases were identified, screening was offered to all residents, with repeat testing 3 months later for HBV-susceptible residents. Molecular testing was performed to assess viral relatedness. Infection control practices were observed. Case-control studies were conducted to evaluate associations between exposures and acute HBV infection in each facility. Results. Six acute HBV cases were identified in each SNF. Viral phylogenetic analysis revealed a high degree of HBV relatedness within, but not between, facilities. No evaluated exposures were significantly associated with acute HBV infection in facility A; those associated with infection in facility B (all odds ratios >20) included injections, hospital or emergency room visits, and daily blood glucose monitoring. Observations revealed absence of trained infection control staff at facility A and suboptimal hand hygiene practices during blood glucose monitoring and insulin injections at facility B. Conclusions. These outbreaks underscore the vulnerability of LTCF residents to acute HBV infection, the importance of surveillance and prompt investigation of incident cases, and the need for improved infection control education to prevent transmission

Projections of the current and future disease burden of hepatitis C virus infection in Malaysia

The prevalence of hepatitis C virus (HCV) infection in Malaysia has been estimated at 2.5% of the adult population. Our objective, satisfying one of the directives of the WHO Framework for Global Action on Viral Hepatitis, was to forecast the HCV disease burden in Malaysia using modelling methods.An age-structured multi-state Markov model was developed to simulate the natural history of HCV infection. We tested three historical incidence scenarios that would give rise to the estimated prevalence in 2009, and calculated the incidence of cirrhosis, end-stage liver disease, and death, and disability-adjusted life-years (DALYs) under each scenario, to the year 2039. In the baseline scenario, current antiviral treatment levels were extended from 2014 to the end of the simulation period. To estimate the disease burden averted under current sustained virological response rates and treatment levels, the baseline scenario was compared to a counterfactual scenario in which no past or future treatment is assumed.In the baseline scenario, the projected disease burden for the year 2039 is 94,900 DALYs/year (95% credible interval (CrI): 77,100 to 124,500), with 2,002 (95% CrI: 1340 to 3040) and 540 (95% CrI: 251 to 1,030) individuals predicted to develop decompensated cirrhosis and hepatocellular carcinoma, respectively, in that year. Although current treatment practice is estimated to avert a cumulative total of 2,200 deaths from DC or HCC, a cumulative total of 63,900 HCV-related deaths is projected by 2039.The HCV-related disease burden is already high and is forecast to rise steeply over the coming decades under current levels of antiviral treatment. Increased governmental resources to improve HCV screening and treatment rates and to reduce transmission are essential to address the high projected HCV disease burden in Malaysia

Experimentelle Kaninchensyphilis und das Reticuloendothelialsystem

Bei experimenteller Kaninchensyphilis fuhrte der Verfasser seine Versuche aus, berucksichtigt auf die klinischen Befunde und die Wassermannsche Reaktion des mit Sp. pallida geimpften Kaninchens bei der Splenektomie, bei der Blockierung des Reticuloendothelialsystemes und bei der Einspritzung des Milzextractes. Die Resultate sind die folgenden: 1. Nachdem Spirochaeta pallida dem Kaninchen in den Hoden geimpft wurde, wurde die Blockierung mit Tusche ausgefuhrt. Im Verlaufe der Infektion zeigten sich lokale Befunde und Wassermannsche Reaktion die in jeder Hinsicht denen der Kontrollen glichen. 2. Beim Kaninchen, welches gleichzeizig entmilzt und mit Sp. pallida geimpft wurde, trat der Lokalaffekt etwas fruhzeizig auf und die Wassermannsche Reaktion war kurzdauernd positiv. 3. Falls die Impfung nach der Entmilzung ausgefuhrt war, stimmten die Resultate fast mit den obigen ein. 4. Wenn die Versuchstiere erst geimpft und dann entmilzt wurden, so wurde eine bemerkenswerte oedematose Anschwellung des Hodensacks und ein Kurzdauernder positive Ausfall der Wa. R. beobachtet. 5. Wenn die Entmilzung, Impfung und Blockierung gleichzeizig ausgefuhrt wurden, so zeigten sich die lokalen sowie metastatischen Befunde bei Versuchstieren wie ohne Entmilzung, doch war die positive Phase der Wa. R. bedeutend verkurzt und dabei ihre Grad niederwertig. 6. Erst geimpft und dann mit Milzextract wiederhohlt gespritzt, so verliefen die lokalen sowie metastatischen Befunde leichter und die Wa. R. brach fruhzeizig positiv aus und dauerte langer. (Autoreferat.

HIV Infection Linked to Injection Use of Oxymorphone in Indiana, 2014-2015

BACKGROUND: In January 2015, a total of 11 new diagnoses of human immunodeficiency virus (HIV) infection were reported in a small community in Indiana. We investigated the extent and cause of the outbreak and implemented control measures. METHODS: We identified an outbreak-related case as laboratory-confirmed HIV infection newly diagnosed after October 1, 2014, in a person who either resided in Scott County, Indiana, or was named by another case patient as a syringe-sharing or sexual partner. HIV polymerase (pol) sequences from case patients were phylogenetically analyzed, and potential risk factors associated with HIV infection were ascertained. RESULTS: From November 18, 2014, to November 1, 2015, HIV infection was diagnosed in 181 case patients. Most of these patients (87.8%) reported having injected the extended-release formulation of the prescription opioid oxymorphone, and 92.3% were coinfected with hepatitis C virus. Among 159 case patients who had an HIV type 1 pol gene sequence, 157 (98.7%) had sequences that were highly related, as determined by phylogenetic analyses. Contact tracing investigations led to the identification of 536 persons who were named as contacts of case patients; 468 of these contacts (87.3%) were located, assessed for risk, tested for HIV, and, if infected, linked to care. The number of times a contact was named as a syringe-sharing partner by a case patient was significantly associated with the risk of HIV infection (adjusted risk ratio for each time named, 1.9; P<0.001). In response to this outbreak, a public health emergency was declared on March 26, 2015, and a syringe-service program in Indiana was established for the first time. CONCLUSIONS: Injection-drug use of extended-release oxymorphone within a network of persons who inject drugs in Indiana led to the introduction and rapid transmission of HIV. (Funded by the state government of Indiana and others.)

Efficient error correction for next-generation sequencing of viral amplicons

<p>Abstract</p> <p>Background</p> <p>Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing.</p> <p>Results</p> <p>In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones.</p> <p>Conclusions</p> <p>Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses.</p> <p>The implementations of the algorithms and data sets used for their testing are available at: <url>http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm</url></p

Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings