Low Temperature and Low UV Indexes Correlated with Peaks of Influenza Virus Activity in Northern Europe during 2010–2018

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010–2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region

Low Temperature and Low UV Indexes Correlated with Peaks of Influenza Virus Activity in Northern Europe during 2010–2018

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010–2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region

Novel activities of safe-in-human broad-spectrum antiviral agents

Abstract According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.Peer reviewe

No detection of macrolide-resistant Mycoplasma pneumoniae from Swedish patients, 1996–2013

Background: Mycoplasma pneumoniae is a common cause of respiratory infections which can cause life-threatening pneumonia and serious extrapulmonary manifestations. Since the year 2000, the emergence of macrolide-resistant M. pneumoniae strains has increased with varying incidences across countries. In China more than 90% of the strains are resistant. M. pneumoniae diagnostics is mostly done with molecular methods, and in Sweden antibiotic resistance surveillance is not routinely performed. The prevalence of macrolide-resistant M. pneumoniae has not previously been studied in Sweden. Material and methods: A total of 563 M. pneumoniae–positive respiratory samples, collected from four counties in Sweden between 1996 and 2013, were screened for mutations associated with macrolide resistance using a duplex FRET real-time PCR method. The real-time PCR targets the 23S rRNA gene, and differentiation between wild-type and resistant strains was achieved with a melting curve analysis. Results: Of the 563 samples included, 548 were analyzed for mutations associated with macrolide resistance. No mutations were found. The detection rate of macrolide-resistant M. pneumoniae in this study was 0% [0.00–0.84%]. Conclusion: No macrolide-resistant M. pneumoniae has been detected in Sweden. However, the emergence and spread of macrolide-resistant M. pneumoniae strains in many countries commands continuous epidemiological surveillance

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden

Molecular typing of Mycoplasma pneumoniae strains in Sweden, 1996–2017, and the emergence of a new P1 cytadhesin gene, Variant 2e

Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains' antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden's population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden

Determination of Hepatitis C (HCV) Genotypes and Drug Resistances By a Efficient and Cost-effective Sequence Analysis Method : One cDNA synthesis, two assays

A more efficient, high specific and cost-effective RT-PCR sequencing method has developed for a correct HCV genotype and study the natural genetic variability and drug resistance within HCV non-structure region. Infection with hepatitis C virus (HCV) frequently leads to chronic hepatitis with an increased risk for the development of liver cirrhosis and liver cancer. HCV is classified into eleven major (designated 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains based on the sequence heterogeneity. In Sweden, the genotype distribution was different from that in studies from other parts of the world, with a lower frequency of genotype 1b and a higher frequency of genotype 1a and 3a. HCVgenotype differences affect responses to antiviral therapy, for exemple, patient infected with genotype 1 responds only 50% to PEG-IFN-á and ribavirin treatment in 48 weeks and approximately 80% of patients infected with HCV genotypes 2 and 3 treated with PEG-IFN-á plus ribavirin in 24 weeks achieve a sustained virological response. It has been suggest that the therapeutic strategy should be different for genotype 1 (and 4-6) and genotypes 2 and 3, respectively. Therefore, determination of HCV genotype and antiviral resistance are important and must be performed after the diagnosis of chronic hepatitis C, in order to provide the most effective treatment for HCV infected patients. To identify a correct genotypes and mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a "One cDNA synthesis and two assays" RT-nestPCR method where we sequenced the HCV NS5b region for the genotyping and protease gene for determination of resistance mutations. cDNA was synthesis using random primer and PCR primers were designed from the NS5b region for genotyping and NS3 regions for determining mutations which covers known 10 protease resistance in NS3, including R155K and V36M. Sequences were then analyzed and phylogenetic tree was made for genotypes according to alignment, and identification of resistance substitutions in the NS3 protease was performed by Seqscape software. RT-nestPCR assay was successful in samples containing >100IU/mL HCV RNA. The accuracy of this method has been validated by QCMD (Quality Control for Molecular Diagnosis, UK). Our method represents a more efficient in identifying mixture of genotypes (2k/1b, 2a/2c/2i), specific and reliable method for differentiation between all genotypes and subtypes, economic and is useful in study natural genetic, mutations and polymorphism within HCV NS3 protease region. This simple, more efficient, specific, low-cost and reproducible method can be used as a routine diagnostic and should also be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden

Simultaneous Detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by Use of Molecular Beacons in a Duplex Real-Time PCR▿

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR

Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses