Reverse Transcription-nested Polymerase Chain Research Articles

Human enteric viruses’ detection in mussels (<i>Mytilus galloprovincialis</i>) farmed in the central Adriatic Sea

Human enteric viruses, such as hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus genogroups I and II (NoVGI and NoVGII), cause infections, and it has been largely demonstrated that mussels play an important role if consumed as raw or undercooked food matrices. This study aimed to investigate, through qualitative and quantitative biomolecular assays, the detection of partial genomic regions belonging to the most relevant enteropathogenic viruses for humans (HAV, HEV, NoVGI and NoVGII) in mussels (Mytilus galloprovincialis) farmed along the coasts of two Italian regions on the central Adriatic Sea: Abruzzo (Casalbordino, Chieti) and Molise (Termoli, Campobasso). A total of 425 animals were sampled, and the respective georeferentiations were registered. A total of 85 pools, each composed of five subjects/aliquot, were formed (22 from Abruzzo and 63 from Molise regions). This step was followed by homogenization and RNA extraction, and then the biomolecular assays [nested reverse transcription polymerase chain reaction (PCR) and real-time reverse transcription-quantitative PCR] were performed. 1.17% of the pool was positive for HAV RNA detection (102 copies/mL), 9.41% for HEV (102-103 copies/mL), 2.35% for NoVGI (101 copies/mL), and no pool was positive for NoVGII. This study demonstrated the human enteric viruses’ presence in mussels farmed in a low-investigated marine area. Based on a one-health point of view, this paper wants to enforce the importance of biomolecular and epidemiological screenings as surveillance systems to guarantee human, animal, and environmental health.

Viral hepatitis E in pregnant women in Belarus: clinical examples and molecular genetic characterization of the virus

Objective. To conduct a clinical and molecular genetic study of two cases of acute hepatitis E in pregnant women from Belarus.Materials and methods. A total of 227 pregnant women with clinical symptoms of liver damage were included in the study. Blood serum samples were used for detection of antibodies to hepatitis E virus by enzyme-linked immunosorbent assay, virus nucleic acid was detected from fecal samples by reverse transcription nested polymerase chain reaction. The nucleotide sequence was determined on an automated sequencer using the Sanger method. Nucleotide sequences were analyzed, genotyped and evolutionary distances calculated using MEGA X software.Results. Out of 227 sera tested, immunoglobulins of class G to hepatitis E virus were detected in 15, which is 6.61%. M immunoglobulins were also detected in the blood of six pregnant women, which is 2.6%. Two clinically evident cases of acute hepatitis E were identified. One of the two patients ended her pregnancy with antenatal fetal death. Virus nucleic acid was isolated from another woman diagnosed with hepatitis E, who had an episode of nutritional contact with raw minced pork. The virus RNA sequence isolated from this patient’s body is clustered into a common phylogenetic branch with the sequence obtained from the body of a patient from Belarus with a history of kidney transplantation and sequences isolated from the body of a domestic pig in the Republic of Belarus.Conclusion. Hepatitis E virus poses a significant threat to pregnant women. A set of preventive measures against hepatitis E in pregnant women has been proposed.

Measurable residual disease study through three different methods can anticipate relapse and guide early interventions in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Measurable residual disease (MRD, previously named minimal residual disease) study can guide therapy adjustments or preemptive interventions that might avoid hematological relapse. Clinical decision making and patient outcome were evaluated in 80 real-life childhood ALL patients, according to the results observed in 544 bone marrow samples analyzed with three MRD methods: multiparametric flow cytometry (MFC), fluorescent in-situ hybridization (FISH) on B or T-purified lymphocytes and patient-specific nested reverse transcription polymerase chain reaction (RT-PCR). Estimated 5year overall survival and event-free survival were 94% and 84.1%, respectively. A total of 12 relapses in 7 patients were associated with positive MRD detection with at least one of the three methods: MFC (p < 0.00001), FISH (p < 0.00001) and RT-PCR (p = 0.013). MRD assessment allowed the anticipation of relapse and adapted early interventions with different approaches including chemotherapy intensification, blinatumomab, HSCT and targeted therapy to halt relapse in five patients, although two of them relapsed afterwards. MFC, FISH and RT-PCR are complementary methods for MRD monitoring in pediatric ALL. Although, our data clearly show that MDR positive detection is associated with relapse, continuation of standard treatment, intensification or other early interventions were able to halt relapse in patients with different risks and genetic background. More sensitive and specific methods are warranted to enhance this approach. However, whether early treatment of MRD can improve overall survival in patients with childhood ALL needs to be evaluated in adequately controlled clinical trials.

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of &lt;i&gt;Aichivirus&lt;/i&gt; A Monitoring in Groundwater

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of &lt;i&gt;Aichivirus&lt;/i&gt; A Monitoring in Groundwater

Comparison of RT-PCR, RT-nested PCRs, and real-time PCR for diagnosis of severe fever with thrombocytopenia syndrome: a prospective study

We designed a highly sensitive reverse transcription nested polymerase chain reaction targeting the M-segment (NPCR-M) of severe fever with thrombocytopenia syndrome (SFTS) virus. NPCR-M was performed in parallel with three other referenced PCR assays QPCR-S, PCR-M, and NPCR-S to assess their clinical usefulness as routine diagnostic techniques for SFTS. In this multi-centered prospective study, 122 blood samples from 38 laboratory-confirmed SFTS patients and 85 control samples were used. The results demonstrated that QPCR-S and NPCR-S had better sensitivity rate up to 21 days after symptom onset however, the PCR-M showed poor sensitivity after 7 days of symptom onset. Our designed NPCR-M had a higher detection rate up to 40 days from symptom onset and revealed the persistence of SFTSV RNA in the early convalescent phase. No false-positive results were seen for the control samples. Additionally, NPCR-M showed positive results for a sample that initially showed negative results from other PCRs and for many other samples collected in the convalescent phase of SFTS. Our designed nested PCR is suitable for SFTSV detection in patients’ blood collected in the acute and early convalescent phase of SFTS, and shows better sensitivity and high specificity even up to 40 days after symptom onset.

First Molecular Evidence for Bat Betacoronavirus in Mindanao

Coronaviruses (CoVs) are common agents of respiratory and gastrointestinal tract infections but are also considered as important emerging infectious disease (EID) agents that have led to global epidemics of severe respiratory disease. Human CoV EIDs have been proposed to have originated from bat CoVs (BtCoV). This study reports the detection of CoV in bats from the University of the Philippines (UP) Mindanao, Davao City using an optimized reverse transcription nested polymerase chain reaction (RT-nPCR) for bat CoV RNA-dependent RNA polymerase (RdRp) gene. Out of 49 bats, only one was positive for the virus (2.04% detection rate). Phylogenetic analysis demonstrated the genetic relatedness of the CoV, which was detected in the fruit bat Macroglossus minimus, to other bat and mammalian CoVs of betacoronavirus under sub-group 2d. The CoV also exhibited 94.55% identity and close genetic relationship with BtCoV 2265/Philippines/2010 from Luzon, Philippines, suggesting a common lineage of these viruses that may have pre-existed prior to biogeographic movements of bats in the country. This is the first reported detection of CoV in bats from Mindanao, hence further surveillance of circulating viruses in wildlife is recommended to expand the understanding of BtCoV evolution and potential for zoonosis. These findings also emphasize the need to limit potential zoonotic transmissions through bat-animal or human interactions by preserving natural habitats.

Oropouche Virus Detection in Febrile Patients' Saliva and Urine Samples in Salvador, Bahia, Brazil.

Oropouche virus (OROV) is a negative-sense, single-stranded RNA arbovirus transmitted to humans by the midge Culicoides paraenesis, causing Oropouche fever. Reports of its outbreak in Brazil have so far been restricted to the Central-Northern region of the country. However, its incidence is underestimated, mainly due to its clinical similarities with other arbovirus diseases, including dengue (DENV), chikungunya (CHIKV), and zika (ZIKV), and the lack of specific diagnostic tests. Here, we report for the first time, the detection of OROV in saliva and urine samples, and cases of autochthone OROV infections in Salvador Metropolitan region, Bahia, a Northeastern capital in the coast of Brazil. Serum, saliva, and urine samples negative for DENV, CHIKV, and ZIKV were tested for OROV using a reverse transcription nested polymerase chain reaction (RT-nested-PCR) protocol, and 2 serum, 2 saliva, and 1 urine samples were positive. This report shows the need for an efficient surveillance system for controlling the spread of this virus, and suggests the use of saliva and urine as alternative samples for OROV detection in the absence of serum samples.

Detection of Hepatitis E Virus in Raw Pork and Pig Viscera As Food in Hebei Province of China.

Hepatitis E virus (HEV) is an important human pathogen with pigs serving as the main natural animal reservoir. In China, pork is the most popular meat, while pig viscera are also widely consumed. The aim of this study was to evaluate the prevalence of HEV among pigs at slaughter, and assess the presence of HEV in raw pork and pig viscera as food. Samples of pig blood, raw pork, liver, kidney, and blood curd were collected from slaughterhouse or (and) retail market. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay based on an ORF2 antigen sandwich kit. HEV RNA was tested by reverse transcription nested polymerase chain reaction (RT-nested PCR) and the viral load was further assessed using quantitative real-time PCR. The final amplicons of RT-nested PCR were sequenced and undergone phylogenetic analysis. Prevalence of antibodies to HEV was 90.4% (104/115) in pigs at slaughtered level, and one serum sample was HEV RNA positive (0.9%, 1/115). HEV RNA was detected in liver, kidney, and blood samples with positivity of 6.1% (7/114), 3.1% (4/129), and 1.2% (2/170) respectively with viral loads ranged 102.4-104.4 (2.4Log-4.4Log) genome equivalents per gram, but not in pork. The HEV RNA prevalence in both liver and kidney were statistically higher than in pork. Phylogenetic analysis showed that all obtained sequences belonged to HEV genotype 4, which were divided into subtypes 4a, 4b, 4d, and 4i, highly identical to the known human and swine HEV sequences in China. The results indicate that raw pig viscera are more likely to harbor HEV than pork, suggesting a higher transmission risk related to consuming pig organs.

Occult Hepatitis C Virus in Blood Donors in Damietta - Egypt

Egypt maintains the highest prevalence of hepatitis C virus infection, affecting an estimated 15%–20% of the population, new cases reported, so we search about uncommon source of hepatitis C virus infection. Occult hepatitis C Virus infection can be detected and founded in the overall public obviously haven’t any diseases, Detection of viral replication in PBMCs may increase spread and transmission of hepatitis C virus during blood transfusion, hemodialysis, and made liver disease development in occult Hepatitis C Virus infected individual. The present study aimed to detect the occult hepatitis C virus infection in healthy blood donors from Damietta-Egypt, And detection of Hepatitis C Virus core antigen. One hundred and fifty blood donors from El-Azhar University Hospital blood bank in New Damietta City were used into the present study. Detection of Hepatitis C Virus RNA in Peripheral blood mononuclear (PBMCs) and plasma by reverse transcription nested polymerase chain reaction (RT-PCR), and also detection of hepatitis C virus core antigen from plasma by ELISA. We founded that: Detection of hepatitis C virus RNA by nested PCR from PBMC among studied donors are eleven (7.9%) on the other hand Hepatitis C Virus RNA in plasma are two (1.4%) so occult C virus are 9/140 (6.4%). By comparison between results of Hepatitis C Virus core Ag and PCR evidence sensitivity 27%, specificity 98.4% in PBMC and sensitivity 100%, specificity 97.8% in Plasma.

Prevalence of severe fever with thrombocytopenia syndrome virus in black goats (Capra hircus coreanae) in the Republic of Korea

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen in China, Japan, and the Republic of Korea (ROK). The aim of this study was to investigate the prevalence of SFTSV antigens and anti-SFTSV antibodies in black goats (Capra hircus coreanae) throughout the ROK. Sera were collected from 737 black goats in nine provinces in the ROK. Eighteen of 737 (2.4%) goat sera were positive for SFTSV on one-step reverse transcription nested polymerase chain reaction. The amplified 346-bp S segments of SFTSV sequences were classified into three genotypes (BG1, BG2, and BG3), and were included in the Japanese clade rather than the Chinese clade, based on phylogenetic analysis. Forty-three of 624 (6.9%) serum samples were seropositive for anti-SFTSV antibodies on enzyme-linked immunosorbent assay analysis. This study is the first to examine the molecular prevalence of SFTSV in goats and the first to perform serological detection of anti-SFTSV antibodies in livestock in the ROK. Moreover, the results indicate that SFTSV is widely distributed in goats and that additional monitoring for SFTSV is needed in livestock in the ROK.

Prevalence and Clinical Impact of Human Pegivirus-1 Infection in HIV-1-Infected Individuals in Yunnan, China.

Human Pegivirus-1 (HPgV-1) may have a beneficial impact on disease progression in human immunodeficiency virus-1 (HIV-1) infection. However, analysis of the genotypic diversity of HPgV-1 and its relevance to the progression of HIV-1 disease remains limited. A total of 1062 HIV-1-infected individuals were recruited in all sixteen prefectures of Yunnan province, China. The reverse transcription nested polymerase chain reaction (RT-nPCR), phylogenetic analyses, and clinical data analyses were used to detect HPgV-1 infection, determine genotype, and analyze HPgV-1 genotype impact on HIV-1 disease progression. The overall positive rate of HPgV-1 RNA was 23.4% (248/1062), and the frequency of HPgV-1 infection in injecting drug users (IDUs) (28.5%, 131/460) was significantly higher than in heterosexuals (19.4%, 117/602). Multiple genotypes were identified in 212 subjects with successful sequencing for the E2 gene, including genotype 7 (55.7%), genotype 3 (34.9%), genotype 4 (4.7%), genotype 2 (3.3%), and an unclassified group (1.4%). Moreover, genotype 7 predominated in IDUs, whereas genotype 3 was the most common in heterosexuals. Our results revealed that HPgV-1 genotype 7 groups exhibited significantly lower HIV-1 viral load and higher CD4+ cell counts. This finding suggests that HPgV-1 genotype 7 may be associated with a better progression of HIV-1 disease.

Prevalence of Rotavirus in Under 5 Year Old Children Hospitalized for Acute Gastroenteritis, Swaziland, 2013 - 2014

<i>Background:</i> Rotavirus is the most common cause of severe diarrhoea among children < 5 years of age worldwide. To determine the prevalence of severe rotavirus infection in children admitted with acute gastroenteritis attending Mbabane Government Referral Hospital in the Hhohho region and Raleigh Fitkin Memorial (RFM) Hospital in the Manzini Region of Swaziland, sentinel surveillance was conducted in January 2013 – December 2014. <i>Methods:</i> This is hospital based sentinel surveillance study. A total of 331 children under the age of five years with acute diarrhoea that were admitted to the hospitals were enrolled into the sentinel surveillance and 302 cases had stool samples tested for rotavirus antigens by enzyme immunoassay ProSpec T Rotavirus kit. The G and P genotypes were established by multiplex nested reverse transcription polymerase chain reaction. <i>Results:</i> Rotavirus was detected in 159 (52.6%) of the 302 children with acute diarrhoea that had stool specimens collected and 108 (68%) of positive cases of rotavirus gastroenteritis were between 6-11 months of age and 91% were below the age of two years. Rotavirus infection occurred with peaks between June - August. During the 2013 – 2014 period, 146 samples were subjected for reverse transcription polymerase chain reaction and genotyping assays. The most common genotypes detected in Swaziland were 82% G2P [4] in 2013 and 93% G1P [8] in 2014. <i>Conclusion:</i> Swaziland would benefit by introducing rotavirus vaccine and hence reduce the hospitalization burden of managing acute diarrhoea cases attributed to Rotavirus. The data from the sentinel hospitals can also be used to monitor the effectiveness of the vaccine once introduced. 1. National Reference Laboratory, Ministry of health, Swaziland- MOH 2. World Health Organization, Inter country support team for East and Southern Africa, Zimbabwe, Harare, 3. Ministry of health, Swaziland 4. World Health Organization, Swaziland 5. Mbabane Government Hospital, Swaziland 6. Raleigh Fitkin Memorial Hospital, Swaziland

Accurate, simple, and inexpensive assays to diagnose F8 gene inversion mutations in hemophilia A patients and carriers

AbstractThe most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based “inverse-shifting” PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect F8 inversion mutations via same-day testing of patient samples.

The Superiority of Allogeneic Hematopoietic Stem Cell Transplantation Over Chemotherapy Alone in the Treatment of Acute Myeloid Leukemia Patients with Mixed Lineage Leukemia (MLL) Rearrangements.

BackgroundAcute myeloid leukemia (AML) patients with mixed lineage leukemia (MLL) gene rearrangements always had a very poor prognosis. In this study, we report the incidence of MLL rearrangements in AML patients using gene analysis, as well as the clinical significance and prognostic features of these rearrangements.Material/MethodsThis retrospective study took place from April 2008 to November 2011 in the People’s Liberation Army General Hospital. A total 433 AML patients were screened by multiple nested reverse transcription polymerase chain reaction (RT-PCR) to determine the incidence of the 11 MLL gene rearrangements. There were 68 cases of MLL gene rearrangements, for a positive rate of 15.7%. A total of 24 patients underwent allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and 34 patients received at least 4 cycles of chemotherapy. Ten patients were lost to follow-up.ResultsThe median follow-up was 29 months. The complete remission (CR) rate was 85.4%. The overall survival (OS) was 57.4±5.9 months for the Allo-HSCT group and 21.0±2.1 months for the chemotherapy group. The Allo-HSCT group had superior survival compared with the chemotherapy group (5-year OS: 59±17% vs. 13±8%, P<0.01; 5-year disease-free survival [DFS]: 65±10% vs. 40±16%, P>0.05). Multivariate analysis showed that transplantation, platelets >50×109/L at onset, and CR are associated with a better OS in MLL rearranged AML patients. Patients with thrombocytopenia and extramedullary involvement were prone to relapse.ConclusionsOur results suggest that Allo-HSCT is superior to chemotherapy alone for treating MLL rearranged AML patients. Patients treated with Allo-HSCT have a better prognosis and a longer survival. CR is an independent prognostic factor for OS, and extramedullary involvement is an independent prognostic factor for DFS. MLL rearranged AML patients with thrombocytopenia at onset <50×109 had very bad OS and DFS.

Prevalence of Occult Hepatitis C Virus in Blood Donors in Zagazig City Blood Banks

Background: Egypt is reported as the country of highest incidence of HCV worldwide. The high rate of new cases reported require investigating uncommon sources of infection. Objectives: The study aimed to investigate the prevalence of occult hepatitis C infection (OCI) among blood donors from Zagazig city blood banks as a probable hidden source of transmission; and to evaluate HCV core Ag detection as a method for diagnosis of occult hepatitis C infection. Methodology: One hundred and forty blood donors from Zagazig university blood bank were incorporated into this study. Reverse transcription nested polymerase chain reaction from Peripheral Blood Monocyte (PBMC) and plasma obtained from subjects was done using HCV specific primers and HCV core antigen was detected using commercial ELISA kit. Results & Conclusion: HCV RNA was detected in PBMC extracted from 10 subjects (7.1 %). Two of them were also positive in plasma (1.4%). The overall incidence of OCI was 8/140 (5.7%). Out of them only one was also identified by ELISA HCV core antigen detection kit. The Comparison between ELISA technique for detection of HCV core antigen and nested PCR revealed sensitivity 30%, specificity 99.2% in PBMC and sensitivity 100%, specificity 98.6% in Plasma.

Epidemiological characteristics and therapeutic strategies of hepatitis C virus genotype 6 in Guangxi area

Objective To investigate the epidemiological characteristics and therapeutic strategies of patients infected with hepatitis C virus (HCV) genotype 6 in Guangxi area. Methods Serum samples were collected from 150 patients with serologic HCV RNA positive in Guangxi, China. Reverse transcription nested polymerase chain reaction (PCR) was employed to amplify HCV NS5B fragments and the DNA products were sequenced. The sequences obtained were compared with the sequences deposited in GenBank to construct a phylogenetic tree. Among the patients who accomplished 48-week treatment of interferon plus ribavirin and 24-week follow-up after stopping medication, 10 cases were infected with genotype 6a and 28 cases with genotype 1 HCV. The virological responses were evaluated at week 4, week 12, week 24 of treatment and week 24 after the end of the treatment. Results Among all recruited 150 cases, 21 (14.0%) cases were HCV genotype 6 including two subtypes 6a (n=20) and 6d (n=1). Genotype 6 HCV mainly affected intravenous drug users, especially with age of ≤40 years old. Phylogenetic tree showed that there was very close evolutionary distance between HCV 6 strains of Guangxi and Hongkong, China strains (Y12083, DQ 480515) and Vietnam strain (EU246930). All of 10 HCV genotype 6a patients who completed 48 weeks of antiviral therapy achieved sustained virological response (SVR). The rate of SVR was higher than that of genotype 1 patients, but without statistically different significance (10/10 vs 75.0%, P>0.05). Conclusion HCV genotype 6 in Guangxi area mainly affects young intravenous drug users with age of ≤40 years old, which has high homology with Hongkong, China and Vietnam standard strains. Patients with HCV 6 genotype infection treated with interferon plus ribavirin for 48 weeks usually achieve favorable SVR. Key words: Guangxi region; Hepacivirus; HCV genotype 6; Phylogenetic tree; Characteristics; Therapy

Mutations in the NS5A gene of hepatitis C virus subtype 1b and response to peg-IFNα-2a/RBV combination therapy in Azerbaijani patients.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease throughout the world. The presence of mutations in different regions of the HCV subtype 1b (HCV-1b) nonstructural 5A (NS5A) gene may be associated with response to interferon therapy. This study evaluated whether amino acid substitutions in the NS5A protein of HCV-1b correlated with response to pegylated interferon alfa-2a (peg-IFNα-2a) and ribavirin (RBV) combination therapy in Azerbaijani patients. From March 2010 to April 2014, a total of 34 chronically HCV-1b-infected Azerbaijani patients were enrolled in this prospective study. After extraction of RNA from plasma specimens, the entire sequences of the NS5A gene of HCV was amplified by reverse transcription nested polymerase chain reaction (RT-nested PCR), and the PCR products were sequenced subsequently. The data that were obtained revealed that there was no correlation between the response to HCV combination therapy and the number of mutations in the NS5A-PKRBD, NS5A-ISDR, and NS5A-V3 regions of HCV. It also was found that changes from isoleucine to valine (I2252 V), aspartic acid to glutamic acid (D2257), arginine to lysine (R2269 K), and arginine to glycine in NS5A-PKRBD and from glycine to glutamic acid (G2379E) in the NS5A-V3 region were not associated with HCV treatment outcome. This study showed that genetic variability in the NS5A-PKRBD, NS5A-ISDR, and NS5A-V3 regions is not a predictive factor of SVR, NR or relapse in HCV genotype1b treated with peg-IFNα-2a/RBV combination therapy.

Transmission of hepatitis C virus among intravenous drug users in the Uppsala region of Sweden

BackgroundEpidemiology and transmission patterns of hepatitis C virus (HCV) are important subjects as we enter a new era of treatment with directly acting antivirals (DAAs). The highest prevalence of HCV in developed countries is found among intravenous drug users (IDUs), where unsafe needle sharing practices provide the main route of infection. Efforts to prohibit the continuous spread of HCV among these groups have been initiated by the community services and health care providers. Our goal was to understand how HCV was transmitted among IDUs within a limited population group. We provide a retrospective study (2005–2007) of the HCV transmission patterns in a population of IDUs in the Uppsala region of Sweden.MethodEighty-two serum samples were collected from IDUs in Uppsala County. Our reverse transcription nested polymerase chain reaction (RT-nested PCR) and sequencing method enabled a comprehensive genetic analysis for a broad spectrum of genotypes of two relatively conserved regions, NS5B and NS3, that encodes for the viral polymerase and protease, respectively. HCV RNA in serum samples was amplified and sequenced with in-house primers. Sequence similarities between individuals and subgroups were analyzed with maximum likelihood (ML) phylogenetic trees. Published HCV reference sequences from other geographic regions and countries were also included for clarity.ResultsPhylogenetic analysis was possible for 59 NS5B (72%) and 29 NS3 (35%) sequences from Uppsala patients. Additionally, we also included 15 NS3 sequences from Örebro patients, making a total of 44 NS3 sequences for the analysis. By analyzing the NS3 sequences, two transmission sets were found between the IDUs (>98% sequence identity), with one set consisting of two individuals and another set consisting of three individuals. In addition, the phylogenetic analysis done with our serum samples displayed clusters that distinguished them from the reference sequences.ConclusionOur method seems to enable us to trace the HCV transmission between IDUs. Furthermore, the method is fairly independent of the time of infection because the method uses relatively conserved HCV sequence regions (i.e. NS5B and NS3).

Genetic variation of nucleocapsid genes of porcine epidemic diarrhea virus field strains in China

Porcine epidemic diarrhea virus (PEDV) poses a great threat to the Chinese swine industry. During 2006–2011, 74.0 % (94/127) of samples from 32 swine-raising farms in 15 provinces were positive for PEDV by reverse transcription nested polymerase chain reaction (RT-nested PCR). The sequences of nucleocapsid (N) genes of 32 representative field strains were determined, and the open reading frames (ORFs) of these N genes were 1326 nucleotides long. The N gene sequences were found to be more than 95 % identical to each other. The number of potential phosphorylation sites in the N protein varied from 5 to 12. A phylogenetic tree based on the N genes showed that the 32 Chinese field strains formed three groups.

Testicular involvement during Toscana virus infection: an unusual manifestation?

Toscana virus (TOSV), transmitted by the Phlebotomus genera, is the most common cause of viral meningitis in the Mediterranean area during summer [1]. Generally, the disease is benign and self-limited, although severe complications such as encephalitis and cerebral ischemia are described [2]. Asymptomatic infections and clinical manifestations in the absence of central nervous system involvement are also documented [3, 4]. We describe an unusual manifestation of TOSV infection in a patient residing in central Italy (Siena district, Tuscany). On 4 September 2012, a 29-year-old previously healthy man was admitted to hospital for fever and severe headache lasting 3 days; testicular pain and diarrhea were also reported. On admission, the patient was suffering and alert with an axillary temperature of 37.3 C, pulse rate of 80 beats/min, respiratory rate of 20 breaths/min, and blood pressure of 110/80 mmHg. Physical examination revealed signs of meningeal irritation, in the absence of focal neurological deficits, tenuous maculopapular rash in the trunk and upper limbs, pharyngeal hyperemia, and swelling and tenderness of the right testicle with no other genital abnormalities. The laboratory tests showed a C-reactive protein level of 1.78 mg/dL (normal value \0.5), white blood cell (WBC) count 11,200/mm (neutrophils 70.4 %), aspartate aminotransferase (AST) 24 U/l, alanine aminotransferase (ALT) 44 U/l, and IgG 582 mg/dL. The erythrocyte sedimentation rate, hemoglobin, platelet count, procalcitonin, renal function, amylase, electrolytes, lactate dehydrogenase, creatine phosphokinase, C3, C4, IgA, IgM, and bilirubin were all within the normal limits. Computed tomography (CT) scan of the brain without contrast medium, chest X-ray, and electrocardiography (ECG) were negative. The testicular ultrasound showed minimal right hydrocele. Cerebrospinal fluid (CSF) examination revealed normal glucose level, increased protein levels (161 mg/dL), and pleocytosis (1,745 cells/mm, 61.7 % lymphocytes). The patient was treated with an empirical therapy based on ceftriaxone and intravenous acyclovir. TOSV was detected by specific nested reverse transcription polymerase chain reaction (RT-PCR) in CSF, while bacterial and fungal cultures, as well as results of PCR for picornavirus, HSV-1 and HSV-2, and Mycobacterium tuberculosis were negative in the same specimen. Blood cultures were negative and serology against TOSV showed positivity for IgM and IgG; antibodies against mumps virus were compatible with past infection. The patient gradually improved, with rapid disappearance of the rash and testicular pain; after 7 days, he was discharged in good condition. This case underlines that, in addition to the typical neurological manifestations, TOSV infection may involve other organs [1]. Although cutaneous rash has been reported several times, testicular involvement, as in the patient described here, has only been documented in a single previous report [3]. From a pathogenetic point of view, the concomitance and the rapid disappearance of genital symptoms during the infection seem to suggest, in the absence of other obvious causes, a direct relationship with viral replication.