[show abstract][hide abstract] ABSTRACT: This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.
Archives of Virology 03/2013; · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study presents epidemiological and clinical data on non-sentinel patients considered by physicians as suspected to be infected with pandemic A(H1N1)2009 virus, from whom clinical specimens were sent for testing to the National Influenza Center, NIPH-NIH in Warsaw, Poland. Between April 28, 2009 and August 10, 2010, 988 (15.7%) out of the 6,311 specimens were tested by the National Influenza Center, including 798 from non-sentinel sources and 190 from sentinel influenza surveillance network. The non-sentinel specimens were tested by conventional RT-PCR to detect influenza A and in the case of positive specimens - one-step real-time RT-PCR to detect the pandemic virus A(H1N1)2009. In 145 (18.2%) cases, infections with the pandemic virus were confirmed, with the highest number in patients aged 15-25. In 45% of the confirmed cases, a history of travel to other countries was registered. The most common symptoms were fever ≥38°C (72.7%), cough (50%), sore throat, and myalgia (26.1%). In 40.7% of the swabbed patients, clinical and epidemiological criteria for the novel influenza A(H1N1)2009, set by the European Commission, were met. There were, however, specimens from persons without any reasonable indication for testing for the pandemic virus, specimens collected incorrectly, and documentation without basic information. These weaknesses resulted in unnecessary costs and overload of health care units. An improvement should be achieved in the area of communication between different pandemic players in the future. More attention is also needed to ensure that requirements and recommendations are known and used.
Advances in experimental medicine and biology 01/2013; 756:271-83. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clinicians often do not consider the presence of more than one viral etiologic agent in respiratory infection, and in many cases they order diagnostics for influenza viruses or recently even only for A(H1N1)2009 virus. However, in a substantial number of patients with a respiratory tract disease, co-infection with various viral pathogens has been confirmed. Although the association between the occurrence of co-infection and substantially higher severity of disease is still unclear, a rapid and proper diagnostics of wide spectrum of viral respiratory pathogens reveals an accurate picture of the disease and is essential for appropriate therapeutic management and control of infection. In the present study we reported five cases of multiple respiratory infection in hospitalized immunosuppressed patients: two double infections with influenza virus (IV) type A/respiratory syncytial virus (RSV) type A and IV type A/coronavirus (CoV) OC43, one infection with four viruses - IV type A/RSV type A and B/CoV OC43, and two cases of mixed infections caused by five viral agents - IV type A and B/RSV type A and B/ parainfluenza type 3 or CoV OC43. Each patient had an underlying chronic disease and received immunosuppressive treatment. Despite a low number of tested specimens, our study shows that the inclusions of multiplex PCR methods for diagnostics of respiratory tract infections and the extension of diagnostic strategies by clinicians to detect viruses other than influenza are very important and make a contribution to identifying the true rate of co-infections and their correlation with the clinical symptoms and severity of disease.
Advances in experimental medicine and biology 01/2013; 756:291-301. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to analyze data collected by the SENTINEL influenza surveillance system in Poland in the first post-pandemic season 2010/2011. The results include weeks 35/2010-17/2011. Physicians registered weekly number of influenza-like illnesses and collected specimens. Laboratory tests were done using PCR and/or real-time PCR or immunofluorescence. Laboratories also isolated the influenza virus. Influenza-like illness incidence amounted to 2802.7/100000. Weekly incidence ranged from 11.3/100000 to 232/100000. The most affected group was children 0-4 years. Influenza-like illness peak occurred between weeks 02/2011 and 11/2011. Influenza infections were confirmed in 34.9% of specimens. More cases were caused by influenza A, including A(H1N1)pdm09 than influenza B (59.9% vs. 40.1%). The isolated strains were similar to A/California/7/2009 or B/Brisbane/60/2008. Season 2010/2011 in Poland did not differ from the rest of Europe. Further improvement is necessary, especially in the area of specimen collection at the beginning of an epidemic season and carrying out the isolation of the influenza virus.
Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists 01/2013; 62(1):51-8. · 0.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Children are an important vector for spreading influenza and they are at increased risk for complications. The appropriate diagnosis of influenza may help start early antiviral treatment and may optimize the use of antibiotics and additional laboratory tests. The objective of this study was to describe the influence of rapid influenza detection test (RIDT) on clinical management of children with acute febrile respiratory tract infections. The method consisted of a prospective, open, cohort study conducted in three primary care clinics in Warsaw, Poland, during the epidemic influenza seasons of 2009/2010 and 2010/2011. A total number of 256 children of the age 0-5 years with symptoms of febrile respiratory tract infection were enrolled into the study. A 115 of them were tested with RIDT (BD Directigen EZ FluA + B) and another 141 children, who were not tested, constituted a control group. We found that RIDT gave positive results in 35 (30%) out of the 115 tested children. Antibiotics, additional blood tests and urinalysis were administered more often in the control group compared with the rapid test group (16% vs. 7%; 14% vs. 5%, and 47% vs. 32%, respectively). Chest radiograms were made only in six cases of children from the control group. We conclude that in children with symptoms of acute febrile respiratory tract infection, the rapid influenza detection test provides a rational use of antivirals, reduces an inappropriate use of antibiotics, and decreases a number of additional tests conducted.
Advances in experimental medicine and biology 01/2013; 755:237-41. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trueperella (Arcanobacterium) pyogenes is an opportunistic animal pathogen, which in European bison is associated with different suppurative infections mainly of the urogenital tract. Little is known about the virulence of this bacterium and about the pathogenesis of infections. The main objective of this study was to determine phenotypic properties and virulence genotypes of the twenty-five T. pyogenes strains isolated from lesions in various tissues of free-living European bison. Classical bacteriological methods were used for phenotypic characterization. Genes encoding seven known and putative virulence factors of T. pyogenes were detected by PCR technique. Analysis of 16S rDNA partial sequences was performed to establish phylogenetic relationships of the isolated strains. All isolates showed typical morphological features of T. pyogenes and variable biochemical activity. Most of them displayed a strong positive effect in synergistic CAMP test. For all isolates the 16S rRNA gene partial sequence was identical to that of the T. pyogenes reference strain. All isolates carried the plo and fimA genes, while the nanH, nanP, cbpA, fimC and fimG genes were present in 40, 44, 12, 88 and 24% of the isolates, respectively. The T. pyogenes strains isolated from European bison represented various phenotypes and virulence genotypes, but there was no association between the investigated properties of the bacteria and the type of anatomopathological lesions from which they were isolated. These results indicate that the studied virulence factors of T. pyogenes are not significant determinants of the localization and type of infection caused by this bacterium.
[show abstract][hide abstract] ABSTRACT: Among different laboratory techniques used in influenza surveillance, methods based on molecular biology, such as real-time PCR, play increasingly important role. They allow detection and identification of viruses currently circulating in human population and responsible for infections and diseases. The aim of the study was to develop duplex real-time PCR assay for detection and differentiation of influenza virus subtype A(H1N1) pdm09 and A(H3N2).
Specific primers targeting a highly conservative region ofhaemagglutinin gene and a dual-color TaqMan probes, labeled with fluorophore JOE (560 nm) and quencher BHQ-1 or CalFluor Red 610 (610 nm) and BHQ-2 were designed. The specificity of duplex reaction was evaluated using RNA isolated from reference strains of influenza virus A(H1N1)pdm09, A(H3N2), A(H1N1) and A(H5N1), B and other respiratory pathogens. Sensitivity of the assay was tested using serial dilutions ofplasmid constructs carrying fragment of specific viral HA gene, in range between 10 and 10(5) copies/sample. A number of 116 clinical samples were tested using developed duplex qPCR assay in comparison with the CDC monoplex assay,
Positive duplex qPCR results were obtained only in samples containing RNA of influenza viruses of a specific subtype. The limit of detection (LOD) of developed method amounted up to 27 copies/sample for A(H1N1)pdm09 and up to 37 copies/sample for A(H3N2).
The results show that developed TaqMan-based duplex qPCR assay is a valuable diagnostic tool in influenza virus surveillance for using in direct study of clinical specimens as well as identification of isolated strains of influenza virus.
Medycyna doświadczalna i mikrobiologia 01/2012; 64(2):129-37.
[show abstract][hide abstract] ABSTRACT: Respiratory viruses contribute to significant morbidity and mortality in healthy and immunocompromised individuals and are considered as a significant economic burden in the healthcare system. The similar clinical symptoms in the course of different viral and bacterial respiratory infections make the proper diagnosis difficult. An accurate and prompt diagnostics is crucial for infection control and patient management decisions, especially regarding the use of antibacterial or antiviral therapy and hospitalization. Moreover, the identification of the causative agent eliminates inappropriate use of antibiotics and may reduce the cost of healthcare. A wide variety of diagnostic procedures is applied for the detection of viral agents responsible for respiratory tract infections. For many years, the viral antigen detection and standard isolation technique in cell culture was the main method used in routine diagnostics. However, in recent years the nucleic acid amplification techniques have become widely used and have significantly improved the sensitivity of viral detection in clinical specimens. Molecular diagnostic assays have contributed to revealing high rates of co-infection (multiplex reactions) and allow identification of agents that are difficult to culture. This paper discusses a number of technical aspects of the current most commonly used techniques, their general principles, main benefits and diagnostic value, but also some of their limitations.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 01/2012; 66:452-60.
[show abstract][hide abstract] ABSTRACT: Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.
Journal of clinical microbiology 04/2011; 49(6):2216-21. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: A total number of 1,081,974 cases of influenza and influenza-like illness were registered in Poland in 2009 (incidence 2,835.9 per 100,000 population). It was nearly 5 times more than in 2008. The impact on increase of the number of reported cases have had two factors: the pandemic of influenza caused by virus A(H1N1)v, and increasing of the surveillance sensitivity. 3,177 (0.29%) cases was laboratory confirmed. In the area of particular regions incidence ranged from 805.2 in świetokrzyskie voivodeship to 5,257.9 in warmifisko-mazurskie voivodeship. Nearly 37% of cases were children under 15 years. The incidence in this age group was 6,851.2 (from 2,010.1 in Swietokrzyskie voivodeship, to 13,291.6 in warmińsko-mazurskie voivodeship). The highest reported incidence was observed in age group 5-14 years (7,135.2). To hospitals, mainly for epidemiological reasons, 8,944 people were sent (0.83% all cases). According to Central Statistical Office data, there were 87 death cases, including 8 (9.2%) children in the age of 15. 70.1% of deaths were registered as cases caused by identified influenza virus.
[show abstract][hide abstract] ABSTRACT: Since C. pseudotuberculosis is a facultative intracellular pathogen the aim of this study was focused on evaluating mechanisms that allowed these bacteria to survive in macrophages and determining their influence on induction of cell death. The influence of Corynebacteria on the programmed cell death of macrophages was determined on the basis of induction the autophagy and apoptosis in the cultures of murine macrophage cell lines J774 infected with bacteria. Corynebacterium pseudotuberculosis strains could survive within macrophages more than 48 hours. During that time bacteria were released as a result of the process that lead to death of phagocytes. This property varied among studied strains. There was no increase of microtubule-associated protein I light chain 3 (MAP I LC3) activity in macrophages infected with examined strains comparing with uninfected cultures and cultures treated with autophagy inducer (rapamycin) that served as negative and positive controls, respectively. The study with confocal microscopy did not show the increasing of caspase-3 activity in the infected macrophages and their nucleus did not reveal the fragmentation.
[show abstract][hide abstract] ABSTRACT: Morel's disease caused by Staphylococcus aureus subsp. anaerobius was diagnosed for the first time in Poland in October 2006 in a goat flock. A second infected flock was found two months later. The course of the disease in both flocks was observed for 15-17 months. Clinical manifestation was confined to abscesses located near major superficial lymph nodes, mostly: superficial cervical, subiliac, parotid and mandibular. At necropsy no other lesions were found. The incubation period was estimated at 3 weeks. Clinical signs were seen both in young and adult goats and up to 7 abscesses in one animal were noted. Abscesses tended to persist for 1 to 5 months, then rupture and heal completely. The initial high in-flock point prevalence in both flocks (93.6% and 84.4%) dropped to approximately 10-30% during next 3-4 months. Until the end of the observation period the in-flock point prevalence remained at this level and only single abscesses were observed, mainly in young animals. No influence of the concurrent caprine arthritis encephalitis virus (CAEV) infection on the clinical course of Morel's disease was noticed. It is to be concluded that the clinical course of Morel's disease in a goat flock resembles caseous lymphadenitis (CLA). However, in Morel's disease abscesses occur more frequently in young goats and are located near, not inside, the lymph nodes, as in the case with CLA. Also, the incubation period of Morel's disease seems to be shorter (3 weeks versus 2-6 months in CLA).
[show abstract][hide abstract] ABSTRACT: Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.
Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists 01/2008; 57(2):105-12. · 0.77 Impact Factor