Use of multiplexed tandem PCR to estimate the prevalence and intensity of Theileria orientalis infections in cattle

This study employed a semi-quantitative, multiplexed tandem PCR (MT-PCR) to assess the prevalence and infection intensity of four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis in cattle in Australia. Genomic DNA samples from blood samples (n = 448) collected from 27 to 32 dairy cows from each of 15 dairy herds with a history of recent theileriosis outbreaks (Group 1), and from blood samples available from 24 cows with or without oriental theileriosis (Group 2) were tested using MT-PCR. Results revealed that all four genotypes were present in Group 1 cattle; genotype buffeli had the highest prevalence (80.5%), followed by genotypes ikeda (71.4%), chitose (38.6%) and type 5 (20.3%). Genotype ikeda had the highest average infection intensity in the cattle (relating to 55,277 DNA copies), followed by buffeli, chitose and type 5 (6354–51,648 copies). For Group 2, results indicated that genotype ikeda had a significantly higher average intensity of infection than buffeli in symptomatic cattle (P < 0.001), and symptomatic cattle had a higher intensity of ikeda than asymptomatic cattle (P = 0.004). Future studies should assess the utility of the present MT-PCR assay as a diagnostic and epidemiological tool in other parts of Australasia and the world.     

Distribution of rotavirus genotypes in Dhaka,Bangladesh, 2012–2016: Re-emergence of G3P[8] after over a decade of interval

Group A rotavirus causes a substantial proportion of diarrhoea related deaths worldwide among children under five years. We analyzed rotavirus prevalence and genotypes distribution among patients admitted with diarrhoea at icddr,b hospital in Dhaka during 2012–16. Stool specimens (n = 1110) were collected from diarrhoea patients and tested for RVA antigen using enzyme immunoassay. Rotavirus positive samples were G (VP7) and P (VP4) genotyped by RT-PCR and sanger sequencing. Data on clinical manifestations were collected from icddr,b hospital surveillance system. A total of 351 (32%) patients were positive for rotavirus antigen, about half of those were children under two years old. During the study period, G1P[8] (27%) was the most prevalent strain, followed by G12P[8] (15%) and G9[P4] (9%). Mixed G or P genotypes were identified in a substantial proportion (23%) with few strains of rare combinations such as G1P[4], G1P[6], G2P[6], G2P[8], G9P[6]. The genotypic fluctuation was noteworthy; G12P[8] was the major strain in 2012–14 but sharply decreased in 2015–16 when G1P[8] became the most common strain. G3P[8] re-emerged (17%) in 2016 after 11 years. Since the Government of Bangladesh has planned to include rotavirus vaccine in national immunization programme from 2018, our data will provide baseline information on rotavirus genotypes in the pre-vaccination era to observe the selection pressure on genotypes in the post vaccination epoch.     

Current Status of Acanthamoeba in Iran: A Narrative Review Article

Background:

Free-living amoebae belonging to the genus Acanthamoeba have an environmental distribution. Amoebic keratitis due to these protozoan parasites continue to rise in Iran and worldwide. In Iran, there are various researches regarding both morphological and molecular identification of Acanthamoeba spp. in environmental and clinical samples. However, there is no thorough review about Acanthamoeba genotypes and their distribution in environmental sources such as water, dust and biofilm in Iran. Besides, according to increasing cases of Amoebic keratitis in the region awareness regarding the pathogenic potential of these sight-threatening amoebae is of utmost importance.

Methods:

We conducted a thorough review based on the database sources such as MEDLINE, PubMed and Google scholar. No restrictions were placed on study date, study design or language of publication. We searched all valuable and relevant information considering the occurrence of the Acanthamoeba in both environmental and clinical samples.

Results:

According to our thorough review Acanthamoeba belonging to T4 genotype is the most prevalent type strain in environmental and clinical samples in several regions in Iran and worldwide, however, there are reports regarding Acanthamoeba belonging to other genotypes such as T2, T3, T5, T6 and T11 and the mentioned point could leads us to more researches with the goal of presenting the real genotype dominance of Acanthamoeba and related disease in the country.

Conclusion:

Overall, the present review will focus on present status of genotypes of Acanthamoeba in Iran during recent years.     

Burden of pediatric hepatitis C

Hepatitis C virus(HCV)is a major health burden infecting 170-210 million people worldwide.Additional 3-4millions are newly-infected annually.Prevalence of pediatric infection varies from 0.05%-0.36%in the United States and Europe;up to 1.8%-5.8%in some developing countries.The highest prevalence occurs in Egypt,sub-Saharan Africa,Amazon basin and Mongolia.HCV has been present in some populations for several centuries,notably genotypes 1 and 2 in West Africa.Parenteral anti-schistosomal therapy practiced in the 1960s until the early 1980s had spread HCV infection throughout Egypt.Parenteral acquisition of HCV remains a major route for infection among Egyptian children.Insufficient screening of transfusions,unsterilized injection equipment and re-used needles and syringes continue to be major routes of HCV transmission in developing countries,whereas vertical transmission and adolescent high-risk behaviors(e.g.,injection drug abuse)are the major routes in developed countries.The risk of vertical transmission from an infected mother to her unborn/newborn infant is approximately 5%.Early stages of HCV infection in children do not lead to marked impairment in the quality of life nor to cognitive,behavioral or emotional dysfunction;however,caregiver stress and family system strain may occur.HCV slowly progresses to serious complications as cirrhosis(1%-2%)and hepatocellular carcinoma(HCC)especially in the presence of risk factors as hemolytic anemias,obesity,treated malignancy,and concomitant human immune deficiency and/or hepatitis B virus co-infection.HCV vaccine remains elusive to date.Understanding the immune mechanisms in patients who successfully cleared the infection is essential for vaccine development.The pediatric standard of care treatment consists of pegylated interferon-α2a or b plus ribavirin for 24-48 wk.The new oral direct acting antivirals,approved for adults,need further evaluation in children.Sustained virologic response varies depending on the viral load,genotype,duration of infection,degree of am     

Recognition of core-derived epitopes from a novel HBV-targeted immunotherapeutic by T-cells from patients infected by different viral genotypes

Hepatitis B virus (HBV) infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Current therapies based on nucleos(t)ide analogs or pegylated-interferon-α lead to control of viral replication in most patients but rarely achieve cure. A potential strategy to control chronic hepatitis B is to restore or induce functional anti-HBV T-cell immune responses using HBV-specific immunotherapeutics. However, viral diversity is a challenge to the development of this class of products as HBV genotypes display a sequence diversity of up to 8%. We have developed a novel HBV-targeted immunotherapeutic, TG1050, based on a non-replicative Adenovirus vector encoding a unique and large fusion protein composed of multiple antigenic regions derived from a HBV genotype D sequence. Using peripheral blood mononuclear cells from 23 patients chronically infected by five distinct genotypes (gt A, B, C, D and E) and various sets of peptides encompassing conserved versus divergent regions of HBV core we have measured ability of TG1050 genotype D core-derived peptides to be recognized by T-cells from patients infected by various genotypes. Overall, PBMCs from 78% of genotype B or C- and 100% genotype A or E-infected patients lead to detection of HBV core-specific T-cells recognizing genotype D antigenic domains located both in conserved and variable regions. This proof-of-concept study supports the clinical development of TG1050 in large patient populations independently of infecting genotypes.     

Rotaviruses: Is their surveillance needed?

Rotaviruses, a major cause of gastroenteritis in children worldwide accounts for around 0.5 million deaths annually. Owing to their segmented genome and frequently evolving capability, these display a wide variation in their genotypes. In addition to commonly circulating genotypes (G1, G2, G3, G4, G9, P[4] and P[8]), a number of infrequent genotypes are being continuously reported to infect humans. These viral strains exhibit variation from one geographical setting to another in their distribution. Though the introduction of vaccines (RotaTeq and Rotarix) proved to be very effective in declining rotavirus associated morbidity and mortality, the number of infections remained same. Unusual genotypes significantly contribute to the rotavirus associated diarrhoeal burden, may reduce the efficacy of the vaccines in use and hence vaccinated individuals may not be benefited. Vaccine introduction may bring about a notable impact on the distribution and prevalence of these viruses due to selection pressure. Moreover, there is a sudden emergence of G2 and G3 in Brazil and United States, respectively, during the years 2006–2008 post-vaccination introduction; G9 and G12 became predominant during the years 1986 through 1998 before the vaccine introduction and now are commonly prevalent strains; and disparity in the predominance of strains after introduction of vaccines and their natural fluctuations poses a vital question on the impact of vaccines on rotavirus strain circulation. This interplay between vaccines and rotavirus strains is yet to be explored, but it certainly enforces the need to continuously monitor these changes in strains prevalence in a particular region. Furthermore, these fluctuations should be considered while administration or development of a vaccine, if rotavirus associated mortality is ever to be controlled.     

Hepatitis C Virus Infection in a Tertiary Care Hospital in Mumbai,India: Identification of a Mixed and Novel Genotype

Purpose: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV being a ribonucleic acid virus has considerable sequence variability. Assessment of viral load and genotype is necessary for designing treatment strategies and monitoring for viral resistance among HCV-infected cases. HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. We undertook this study to observe genotype distribution of the virus in HCV patients from Mumbai. Materials and Methods: Between January 2017 and December 2017, the study was conducted on 120 chronic hepatitis outpatients from a tertiary care hospital, Mumbai, after obtaining ethics approval. All these diagnosed cases of HCV were subjected to molecular diagnosis in a research institute, Mumbai, by real-time polymerase chain reaction-based techniques. Results: Males were more preponderant than females with HCV infection, and the highest number of HCV-infected cases was observed in the age group of 41–50 years. Genotype 3 (n = 70; 58.3%) accounted for the highest number of cases followed by genotypes 1b (n = 29; 24.2%) and then 1a (n = 14; 11.7%). Mixed genotypes 1b + 3 and individual genotype 4 were found in two cases each (1.7%). A total of three samples (2.5%) were found with untypeable genotype. Conclusion: The major HCV genotype observed was 3 which is difficult to treat with direct-acting antivirals, owing to the more rapid progression of liver disease, increased rates of steatosis (non-alcoholic fatty liver disease), a higher risk for cancer (HCC). We believe this study is the first one to address the prevalence of mixed genotypes and untypeable genotype from India.     

Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay

BACKGROUND: The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment. METHODS: Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes. RESULTS: The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele. CONCLUSIONS: This PCR assay is a rapid and reliable technique for TLR4 genotyping.