Genotype Subtype Research Articles

Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method

Hepatitis E virus (HEV) is a pathogen of increasing importance, which can be zoonotically transmitted from domestic pigs, wild boar, and deer to humans. Foodborne transmission by consumption of raw and undercooked liver, meat, or sausages prepared from infected animals has been documented. The aim of this study was to investigate the distribution of HEV in different types of sausages sold in Germany. As no standardized methods for HEV detection in food exist, several techniques of sample homogenization, virus concentration and nucleic acid extraction followed by real-time RT-PCR were compared using artificially contaminated sausages. A method using TRI Reagent® Solution showed the best efficacy of matrix disruption and a treatment with chloroform followed by a silica-based RNA extraction method resulted in the highest HEV detection rates. The detection limit of the method was 2.9×103 and 5.3×104 genome equivalents per 5g raw sausage and 2g liver sausage, respectively. Application of the method to raw and liver sausages from retail in Germany resulted in the HEV genome detection in 14 out of 70 (20%) raw sausages and in 11 out of 50 (22%) liver sausages. The detected HEV sequences showed a high diversity and belonged to different subtypes of HEV genotype 3. The results indicate a broad distribution of HEV-RNA in meat products sold in Germany; however, the infectivity of the detected virus remains to be assessed in future.

Resistance to direct-acting antiviral agents: clinical utility and significance.

This article examines the dynamics and factors underlying hepatitis C virus (HCV) resistance, along with their impact on daily clinical management of HCV-infected patients. Across available treatment-regimens, GT-3 is the most difficult-to-cure genotype, but also genotype-1a may show lower success-rates compared with genotype-1b. Natural resistance to NS3, NS5A and NS5B inhibitors may contribute to treatment failures. The Q80K NS3-protease mutation affects sensibility to simeprevir + peg-interferon/ribavirin combinations. It reaches up to 48% prevalence in genotype-1a in some studies (but it is lower in other). Resistant variants (particularly in NS5A) developed at failure can persist, in a substantial proportion of patients, even 3 years after treatment-discontinuation, potentially affecting readministration of the same direct-acting antiviral agent (DAA)-class. This will become an issue for those patients failing all-oral regimens with multiple-resistant viruses. Recent data support the importance of an accurate genotype and genotype-1 subtype (1a/1b) assignment prior therapy. Resistance testing at baseline has no clear indication so far in clinical practice for all-DAA regimens selection, while it remains a valuable option at the retreatment of patients who failed DAA-containing regimens, provided that data are generated to inform treatment decisions based on the results of resistance testing. In this context, long-term RAVs persistence after failure should be taken into account.

Effectiveness of sofosbuvir-based regimens in genotype 1 and 2 hepatitis C virus infection in 4026 U.S. Veterans.

Real-world effectiveness data are needed to inform hepatitis C virus (HCV) treatment decisions. To assess sustained virological response (SVR) of sofosbuvir (SOF)-based regimens in routine medical practice. Observational, intent-to-treat cohort analysis of genotype 1 and 2 HCV-infected veterans initiating SOF-based regimens with recommended treatment duration of 12weeks. Four thousand and twenty-six veterans with genotype 1 (N=3203) and genotype 2 (N=823) comprise the cohort. SVR rates for genotype 1 were 66.8% for SOF+peginterferon+ribavirin (RBV), 75.3% for SOF+simeprevir (SIM), 74.1% for SOF+SIM+RBV and for genotype 2 were 79.0% for SOF+RBV. Genotype 1 patients were less likely to achieve SVR with BMI ≥30 (OR 0.64, 95% CI 0.49-0.84, P<0.001), a history of decompensated liver disease (OR 0.51, 95% CI 0.36-0.71, P<0.001), treatment experience (OR 0.58, 95% CI 0.48-0.71, P<0.001), APRI >2 (OR 0.44, 95% CI 0.36-0.55, P<0.001) and with SOF+PEG+RBV compared with SOF+SIM (OR 0.50, 95% CI 0.40-0.62, P<0.001). Age, sex, race/ethnicity, diabetes and genotype subtype did not predict SVR. Odds of achieving SVR with SOF+SIM+RBV did not differ compared with SOF+SIM (OR 1.03, 95% CI 0.75-1.44, P=0.86). Genotype 2 patients were less likely to achieve SVR with prior treatment experience (OR 0.55, 95% CI 0.35-0.88, P=0.009) and APRI >2 (OR 0.39, 95% CI 0.25-0.62, P<0.001). In this real-world cohort, SVR rates were lower than in clinical trials. Genotype 1 and 2 HCV-infected patients with advanced liver disease by APRI >2 or FIB-4>3.25 were significantly less likely to achieve SVR. For genotype 1, a SOF+SIM±RBV regimen was associated with a higher likelihood of SVR.

Polymorphisms in CYP1B1, CYP3A5, GSTT1, and SULT1A1 Are Associated with Early Age Acute Leukemia.

Based on observational studies, early age leukemia (EAL) was associated with maternal hormone exposure during pregnancy. We studied the association between genetic polymorphisms of estrogen metabolism and EAL. Using data from the Brazilian Collaborative Study Group of Infant Acute Leukemia (2000–2012), 350 cases and 404 age-matched controls and 134 mothers of cases and controls were genotyped to explore polymorphisms in genes of the estrogen metabolism pathway: CYP1B1 (c.1294C>G, rs1056836), CYP3A4 (c.-392A>G, rs2740574), CYP3A5 (c.219-237G>A, rs776746), GSTM1/GSTT1 deletions, and SULT1A1 (c.638G>A, rs9282861; and c.667A>G, rs1801030). Logistic regression was used to calculate the odds ratios (OR) with 95% confidence intervals (CIs), and unconditional logistic regression was used to estimate adjusted odds ratios (aORs) by ethnicity. Because of multiple testing, p values < 0.01 were significant after Bonferroni correction. SULT1A1 (c.638G>A) was associated to infant acute lymphoblastic leukemia and acute myeloid leukemia (AML) risk in males (additive model: aOR = 0.52; 95% CI: 0.29–0.95, p = 0.03; dominant model: aOR = 2.18; 95% CI: 1.17–4.05, p = 0.01, respectively). CYP1B1 polymorphism was associated with a decreased risk of AML either for non-white or female children (additive model: OR = 0.24; 95% CI: 0.08–0.76, p < 0.01; additive model: aOR = 0.27; 95% CI: 0.08–0.89, p = 0.03, respectively). Since polymorphisms of Cytochrome P450 genes presented gender-specific risk associations, we also investigated their expression. CYP1B1 was not expressed in 57.1% of EAL cases, and its expression varied by genotype, gender, and leukemia subtype. Maternal-fetal GSTT1 null genotype was associated with risk of EAL. This study shows that polymorphisms in genes of estrogen metabolism confer genetic susceptibility to EAL, mainly in males, and maternal susceptibility genes modify the risk for developing EAL in newborns.

The prevalence of human T-lymphotropic virus infection among blood donors in southeast China, 2004-2013.

BackgroundThe human T-lymphotropic virus type 1 (HTLV-1) which is associated with the diseases of adult T-cell leukemia/lymphoma, HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP) and HTLV-associated uveitis, can cause transfusion-transmitted infections. Although HTLV screening of blood donors was already routinely performed in developed countries, little is know about the HTLV prevalence among blood donors in developing countries which do not perform HTLV screening, such as China.Objectives &AimsTo systematically characterize the prevalence of HTLV infection among bloods in southeast China.MethodsA 10-year survey for HTLV prevalence in blood donors was performed in Xiamen, southeast China, during 2004-2013. The HTLV-1/2 of blood donations were screened by enzyme-linked immunosorbent assay, following with confirmation by western blot assay and 9nucleic acid testing. The HTLV-1 prevalences in donors from different cities were calculated. Viral sequences derived from identified HTLV-positive cases were sequenced and analyzed.ResultsAmong 253,855 blood donors, 43 were confirmed to be seropositive for HTLV-1 (16.9 per 100,000 95% CI: 12.3-22.8) and none HTLV-2 infection was found. The HTLV-1 prevalence varied significantly in donors from different cities. Donors from cities in Fujian province (24.3 per 100,000, 95%CI: 17.4-33.1) had a significantly higher (p=0.001) HTLV-1 seroprevalence than those who were born in non-Fujian cities (3.4 per 100,000, 95%CI: 0.7-9.8). Among nine cities in Fujian province, the highest prevalence was found in blood donors from Ningde (171.3 per 100,000, 95%CI: 91.3-292.8) which is a coastal city in the northeast of Fujian. Molecular characterization of viral sequences from 27 HTLV-1 carriers revealed 25 were Transcontinental subtype of genotype A and 2 were Japanese subtype of genotype A. Interestingly, 12 of 25 Transcontinental subtype sequences harbored a characteristic L55P mutation in viral gp46 protein, which was only presented in the Transcontinental subtype sequences from Japan and Taiwan but not in that from other countries.ConclusionsAlthough China is considered to be a non-endemic region for HTLV, the HTLV-1 prevalence in blood donors is significantly higher in Fujian province, southeast China. A higher prevalence of HTLV-1 in the Fujian may be attributed to endemic foci in the city of Ningde.

Phenotypic and Genotypic Characterization of Salmonella enterica Serovar Enteritidis Isolates Associated with a Mousse Cake-Related Outbreak of Gastroenteritis in Ningbo, China.

Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is a major pathogen responsible for causing the largest number of sporadic cases and outbreaks of human salmonellosis worldwide. In this study, an outbreak of Salmonella Enteritidis involving 112 cases in Ningbo, China was investigated with a combination of genotypic subtyping methods and phenotypic analysis. The pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis profiles showed that most of the outbreak clinical isolates (22/23) were indistinguishable from each other and were identical to the isolates obtained from implicated mousse cakes, demonstrating that this outbreak of gastroenteritis was caused by Salmonella Enteritidis-contaminated mousse cakes. Moreover, all isolates, irrespective of source, had an identical antibiotic susceptibility pattern. Five virulence-associated genes in Salmonella pathogenicity islands and the plasmid-associated virulence genes spvB/C were present in both the food and clinical isolates. Importantly, all of these isolates can survive well under low-temperature treatment, indicating that manufacturers of foodstuffs with raw ingredients (not subjected to thermal processing) should use an effective approach to prevent or eliminate the microbial hazards to public health.

Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

Recent insights into the genetic diversity, epidemiology and clinical relevance of Blastocystis species.

Blastocystis is a widely prevalent intestinal protozoan of man and other animals. It is associated with gastro intestinal disorders and symptoms. Emerging literature suggests that several subtypes of this parasite exist and are responsible for virulence, pathogenicity and symptoms. A variety of new diagnostic methods have been developed which help to recognize genotypic variations, subtype relationship and determination of carrier state. In symptomatic patients, when the other etiological agents have been excluded, it is prudent to treat Blastocystis infection. Metronidazole remains the drug of choice for treatment. Further research is required to shed light on taxonomy, biology and virulence of this organism.

Molecular characterization of hepatitis C virus genotype 6 subtypes in Thai blood donors.

Hepatitis C virus (HCV) genotype is important for identifying effective antiviral therapy, evaluating pathogenic severity, and tracking transmission routes. In Thailand, HCV genotypes 3 and 1 are the most common. We have previously demonstrated an increasing appearance of genotype 6 in HCV infections in Thailand. However, only limited epidemiological data on genotype 6 in Thailand are available. This study aimed to characterize HCV genotype 6 among apparently healthy Thai blood donors. In total, 240 blood samples were collected from Phitsanulok Regional Blood Center, Phitsanulok, Thailand. RNA was reverse transcribed and amplified by the nested polymerase chain reaction. HCV genotyping was performed by direct sequencing and phylogenetic tree analysis of core sequences. Amino acid polymorphism of various subtypes of HCV genotype 6 was investigated. Of the 240 samples, 192 were successfully sequenced for the core region and 84 were determined to be of HCV genotype 6 by phylogenetic analysis. The most prevalent HCV-6 subtypes were 6f>6n>6c>6i. Amino acid sequences of the partial core region among these four subtypes differed by one to seven residues. For HCV-6, the subtype 6f was commonly found in Thai blood donors. Comparison of core protein from various HCV-6 subtypes showed substantial polymorphisms, which may form the basis of future studies using samples from patients with clear HCV histories. This feature can be applied to therapies tailored to particular genotype variants.

Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping.

Objective:The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey.Methods:One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method.Results:Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b.Conclusion:The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.

Polymorphisms of CYP1B1, CYP3A4, CYP3A5, GSTM1, GSTTT1 and SULT1A1 Associated with Early Age Acute Leukemia

The acute leukemia causation pathway has been postulated to be a multistep process with interaction between environmental and genetic susceptibility factors. Based on previous observation, early age leukemia (EAL) was associated with maternal hormone exposures.The aim of the study was to explore whether polymorphisms in genes of the estrogen-metabolism pathway could be related to EAL. The study included 754 children, being 350 EAL and 404 age-matched controls and 134 mothers of EAL and controls. Polymorphisms of CYP1B1 (rs1056836), CYP3A4 (rs2740574), and CYP3A5 (rs776746) were identified by RFLP; GSTM1 and GSTT1 deletions were detected by multiplex PCR, whilst an internal control (CYP1A1) was co-amplified; SULT1A1 rs9282861 and rs1801030 were genotyped on PyroMark Q24. Gene expression was performed by Real-time PCR using Platinum® SYBR® Green qPCR SuperMix. Logistic regression was used to evaluate the associations adjusted on skin color. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). CYP1B1 and CYP3A5 expression analyzed using Kruskal-Wallis and Dunn's Multiple Comparison tests. GSTT1*0, CYP3A4*1B, and CYP3A5*1 were associated with an increased risk of acute myeloid leukemia (AML) after adjusting for multiple comparisons regarding non-whites and male children (OR 1.91, 95% CI: 1.12, 3.28; OR 2.44, 95% CI: 1.10, 5.43; OR 1.98, 95% CI: 0.96, 4.10, respectively). CYP1B1 was not expressed in 57.1% of EAL cases; expression varied by genotype, gender, and leukemia subtype. AML was associated with distinct modulations: CYP1B1*3/SULT1A1*3 decreased and CYP3A4*1B/SULT1A1*2 increased EAL risk (OR 0.24, 95% CI: 0.10, 0.55; OR 2.06, 95% CI: 0.96, 4.42, respectively). Maternal-fetal genotypes were associated with EAL based on CYP3A4*1B and GSTT1*0. Therefore, genetic polymorphisms involved in estrogen metabolism confer different susceptibilities for EAL, modulating leukemia subtype in males.

An outbreak of extremely drug-resistant Pseudomonas aeruginosa in a tertiary care pediatric hospital in Italy.

BackgroundExtensively drug-resistant Pseudomonas aeruginosa (XDR-PA) isolates are susceptible to only one or two classes of antibiotics. In 2011–2012, we investigated an outbreak of XDR-PA affecting children with onco-hematological diseases.MethodsOutbreak investigation included ascertainment of cases, tracing of intestinal carriers and environmental surveillance. Contact precautions were adopted for patients with infection or colonization. Isolates were tested for antimicrobial susceptibility; phenotypic confirmation of carbapenemase production was performed, and carbapenemase genes were tested by multiplex polymerase-chain-reaction (PCR). Genotypes were determined by pulsed-field gel electrophoresis (PFGE).ResultsXDR-PA was isolated from 27 patients; 12 had bacteremia, 6 other infections and 9 were colonized. Severe neutropenia was significantly associated with bacteremia. Bloodstream-infection mortality rate was 67%. All isolates were resistant to carbapenems, cephalosporins and penicillins + β-lactamase inhibitors. Isolates were susceptible only to colistin in 22 patients, to colistin and amikacin in 4, and to ciprofloxacin and colistin in 1. PFGE results identified 6 subtypes of a single genotype, associated with clusters of cases, and 4 sporadic genotypes. Two sporadic isolates were metallo-β-lactamase producers, negative to PCR. All other isolates were metallo-β-lactamase producers due to the presence of a VIM carbapenemase. Incidence of XDR-PA infections decreased from 0.72 cases/1,000 inpatient-days in March 2011-March 2012, to 0.34/1,000 in April-December 2012, after implementation of active finding of intestinal carriers on all onco-hematological inpatients.ConclusionsControl measures targeting intestinal carriers are crucial in limiting in-hospital transmission of XDR-PA polyclonal strains, protecting more vulnerable patients, such as severely neutropenic children, from developing clinical infections.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-494) contains supplementary material, which is available to authorized users.

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5′UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5′UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5′UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% –100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.

Hepatitis E virus genotype 4 in a pig farm, Italy, 2013.

Zoonotic strains of hepatitis E virus (HEV) in Europe have been reported to belong to genotypes 3 and 4. In 2012 and 2013, 57 pig farms in Northern Italy that had previously resulted seropositive for HEV were surveyed for the presence of the virus, with positive samples subsequently genotyped. Hepatitis E RNA was identified in 17/57 (29·8%) seropositive farms. Phylogenetic analysis demonstrated that distinct subtypes of genotype 3 were circulating in the north-east of Italy; as well, for the first time in the Italian swine population, genotype 4 was identified and attributed to subtype d.

Evaluation of the Abbott RealTime HCV genotype II RUO (GT II) assay with reference to 5′UTR, core and NS5B sequencing

BackgroundHCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. ObjectiveTo evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5′UTR, core and NS5B. Study designThe Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5′UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. ResultsAmong routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5′UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5′UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. ConclusionsThe Abbott assay is an automated HCV genotyping method with improved accuracy over 5′UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method.

Detection of some phenotypic and genotypic characteristics of Staphylococcus aureus isolated from food items in the Czech Republic

Staphylococcus aureus is an important foodborne pathogen and can produce a wide range enterotoxins, which contribute to food poisoning. The aim of this study was to interrogate foodborne strains of S. aureus for phenotypic and genotypic characteristics. Strains were screened for enterotoxins, hemolysins and antimicrobial resistance, and the genetic relationship between strains was described after pulsed-field gel electrophoresis (PFGE) analysis. Of the S. aureus strains, 82.8 % (n = 93) harboured one or more of the following enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej); 39.8 % of strains demonstrated se genes and 43 % carried from two to five se genes, while 17.2 % of the strains possessed none of the genes examined. The most commonly detected toxin genes were sea, seb, sec and seg. The presence of genes coding for antibiotic resistance such as blaZ, vanA, vanB and mecA was investigated by polymerase chain reaction (PCR). Seventy-two strains carrying the blaZ gene exhibited phenotypic resistance to ampicillin and penicillin. Ten strains (10.75 %) carried the mecA gene and correspondingly demonstrated resistance to oxacillin. The presence of vancomycin resistance genes, vanA and vanB, was not detected. Genotypic subtyping was performed using PFGE with SmaI restriction enzyme. The genetic relationships between enterotoxin harboring strains and non-enterotoxigenic strains were explored. Twenty-four different pulsotypes were generated from 93 food isolates with a similarity level of 88 %.

Defining a comprehensive verotype using electronic health records for personalized medicine.

The burgeoning adoption of electronic health records (EHR) introduces a golden opportunity for studying individual manifestations of myriad diseases, which is called 'EHR phenotyping'. In this paper, we break down this concept by: relating it to phenotype definitions from Johannsen; comparing it to cohort identification and disease subtyping; introducing a new concept called 'verotype' (Latin: vere = true, actually) to represent the 'true' population of similar patients for treatment purposes through the integration of genotype, phenotype, and disease subtype (eg, specific glucose value pattern in patients with diabetes) information; analyzing the value of the 'verotype' concept for personalized medicine; and outlining the potential for using network-based approaches to reverse engineer clinical disease subtypes.

Cellular HIV Type 1 DNA Levels Are Equivalent Among Drug-Sensitive and Drug-Resistant Strains in Newly Diagnosed and Antiretroviral Naive Patients

The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5 coreceptor tropism dominance suggests that both drug-resistant and drug-sensitive strains behave similarly in early infection in newly diagnosed patients.

A New Subtype of Hepatitis C Virus Genotype 3: Analysis of Available Evidence

BackgroundHepatitis C virus (HCV) is one of the leading causes of chronic liver disease. Seven genotypes and more than 80 subtypes have been identified for HCV so far. To date, 10 subtypes (3a to 3i; and 3k) of HCV genotype 3 have been identified. In 2006, two HCV isolates were reported from Iran that belonged to a new subtype of genotype 3. However, considering the consensus proposal for HCV genotype nomenclature, the available sequences of the new subtype did not correspond to the regions that are required to be analyzed prior to subtype assignment. During a study on the molecular epidemiology of HCV in Iran, an HCV isolate (FSM165) which seemed to belong to a new subtype of genotype 3 was obtained from a patient residing in Tehran, Iran.ObjectivesThe aim of this study was to assess the relatedness of isolate FM165 together with several sequences retrieved from the database to the new HCV-3 subtype reported from Iran in 2006.Materials and MethodsVarious parts of the genome including the core/E1 region and two segments of the NS5B region were amplified and sequenced for isolate FSM165. Furthermore, using the Basic Local Alignment Search Tool (BLAST), the HCV database was searched for sequences that had a high level of similarity with sequences of FSM165 isolate and such sequences were retrieved from the database. To investigate the relatedness of isolate FSM165 and also the retrieved sequences to a new HCV-3 subtype reported previously, phylogenetic analyses were performed using the Kimura two-parameter model and the neighbor joining method.ResultsPhylogenetic analysis of the partial NS5B region demonstrated the relatedness of isolate FSM165 to the new subtype reported from Iran in 2006. Moreover, some core/E1 and NS5B sequences that had a high level of similarity with FSM165 isolate were found through searching the HCV database. These sequences were previously either misclassified or could not be accurately classified. Phylogenetic analyses showed that all of the described sequences belonged to the new subtype of HCV genotype 3.ConclusionsData suggests that the new subtype has a vast geographical distribution in Iran. The core/E1 and the NS5B sequences described in this paper can be used as references for the new HCV-3 subtype in future studies.

Phylogeny, spatio-temporal phylodynamics and evolutionary scenario of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) in wild boars: Fast dispersal and high genetic diversity

Torque teno sus viruses (TTSuV1-2), members of the newly established family Anelloviridae are considered non-pathogenic emerging viral agents of Suidae species. However, the genetic diversity, phylogeny and evolutionary processes responsible for the emergence and spread of TTSuVs in wild boars remain poorly understood. Here we implemented phylogenetic and evolutionary analyses to address key questions on the genetic diversity, evolutionary scenario and spatio-temporal dynamics of emerging TTSuVs in wild boars of the Western region (Transylvania) of Romania. High levels of genetic diversity of wild boar origin TTSuV1-2 have been found as well as a new TTSuV1 genotype and several new subtypes. Phylogenies suggest that several wild boar viral strains in both TTSuV species are likely to have emerged from a well-defined ancestor approximately 40 (TTSuV1) and 18 (TTSuV2) years ago and showed independent evolutionary trajectories. Bayesian phylogeography showed an intense flow of viral strains throughout the Transylvanian counties possibly related to wild host migrations, facilitating the rapid spread of TTSuVs. The intra-genotype and inter- and intrahost level recombination, intense spatio-temporal viral flow and the positively selected sites found in the ORF2 genes should be considered important driving forces shaping TTSuVs evolution. The first reported rates of nucleotide substitution for porcine anelloviruses, estimated to be 5.29-5.51 × 10(-4)subs site(-1)year(-1), are in line with those measured previously for mammalian ssDNA viruses and RNA viruses. These high evolutionary rates of TTSuVs, independent of recombination, are reflections of adaptive evolution, an important factor in the emergence of novel viral variants which may explain their ability to emerge in Suidae hosts.