A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5′–3′ nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997–1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998–1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997–1998 and 1998–1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.

Annual influenza epidemics are caused by rapid evolution of the viral genome. Continuous and extensive antigenic variation has been shown for hemagglutinin (HA), the principal immunizing antigen of the virus. Monitoring of the antigenicity of circulating influenza viruses is necessary for selection of the most suitable vaccine strains. In this study, characterization of influenza A/H3N2 and influenza B viruses recently circulating in Germany was performed by molecular and antigenic analysis. Sequencing and phylogenetic analysis of the HA1 gene revealed that two distinct groups of H3N2 viruses co-circulated during 1997/1998. The majority of isolates clustered with the new drift variant A/Sydney/5/97, as was also shown by antigenic characterization. A noteworthy genetic drift of H3N2 viruses was evident during the winter 1998/1999. However, serological characterization using hemagglutinin inhibition tests did not result in detection of viruses belonging to different groups as confirmed by molecular analysis. Influenza B viruses isolated during 1996/1997 were antigenically closely related to the prototype vaccine strains B/Beijing/184/93 or B/Harbin/7/94. Molecular analysis demonstrated that our German 1996/1997 isolates differed by nine amino acids from B/Harbin/7/94 and represented a group of viruses that was completely different from the Harbin strain. Retrospective studies revealed the circulation of B/Yamanshi/166/98-like viruses in Germany already during the 1996/1997 season. Our results suggest that molecular analysis of the HA gene is important to complement the antigenic characterization for a better selection of appropriate vaccine strains.

The aim of this serological study was to demonstrate the extent to which antibodies react against subsequent drift variants, after vaccination with split vaccine (Fluarix^TM). Antibody titers have been determined by hemagglutination inhibition test (HI) against different influenza A and B drift variants in sera from three past multicenter trials. Individuals of two different age groups, i.e. 18–60 years and above 60 years, were enrolled. Vaccine components influenza A/H1N1 and influenza B of Fluarix^TM show a high degree of cross immunogenicity against subsequent homologous drift variants. The genetically more variable component influenza A/H3N2 shows somewhat lower protection rates. High levels of cross immunogenicity were found between the variants of influenza A/Panama/2007/99 (H3N2) and influenza A/Wyoming/3/2003 (H3N2). The results demonstrate that in situations where drift variants emerge too late to be included in the influenza vaccine formulation, the cross-protection conferred must be evaluated on a case-by-case basis.

A newly developed colorimetric method, DNA enzyme immunoassay (DEIA), was applied to the detection of neuraminidase subtypes N1 and N2 of influenza A viruses. Reverse transcription and polymerase chain reaction with universal primers were used for genomic amplification of H1N1, H2N2, and H3N2 strains. Following amplification, an aliquot of the PCR product was hybridized to biotinylated DNA sequences (N1/N2 probes) immobilized on microtiter wells. The hybridization event was revealed by monoclonal antibodies to double stranded DNA in a standard ELISA reaction. The assay described here was able to distinguish accurately between the two neuraminidase subtypes of human influenza A viruses. It is a simple and rapid method facilitating the handling of a large number of samples and therefore seems to be easily applicable to diagnostic laboratories.

In the context of influenza pandemic preparedness planning, a surveillance system for influenza and other acute respiratory illnesses was implemented in Lower Saxony at the beginning of the influenza season 2004/2005 and coordinated by the Governmental Institute of Public Health of Lower Saxony. This surveillance system represents an addition to already existing national monitoring systems. The goal of this surveillance system is to have available prompt information on the beginning, course and end of the influenza season and to recognise the spectrum of pathogens and identify outbreaks of other viral acute respiratory illnesses (ARI). For this purpose an all-season surveillance was established consisting of two supplementary modules. The first module is a symptom-oriented surveillance of acute respiratory illnesses in children of pre-school day care facilities. In the second module a virological surveillance in co-operation with selected medical practices was established. While the temporal course and burden of ARI in all Lower Saxony can be assessed by the surveillance of children in the day-care facilities in a sensitive, but less specific way, the virological surveillance provides highly specific information on the prevailing pathogens in ARI patients at a certain time. This information, in return, gives an indication about the responsible pathogens causing ARI in children of the day-care facilities. The first experience with these two complementary surveillance modules shows that in Lower Saxony a well accepted, prompt and meaningful monitoring system is available for the recognition and description of the occurrence of ARI and concomitantly of influenza. An extension of this surveillance to other pathogens or disease scenarios is possible.

The continuous antigenic drift of influenza viruses requires annual adaptation of the vaccine. Protection depends largely on the match of the variants represented in the vaccine with the viruses actually known to be in circulation and may differ considerably from season to season. Therefore studies to assess the efficacy and effectiveness of the vaccine are conducted rather sporadically on an annual basis and it would be desirable to make use of routinely available data from surveillance programs. We compared two different approaches: (1) the "screening method" where cases are identified from laboratory data and controls are taken from data on vaccination rates and (2) a second method that uses the same cases, but controls were influenza-negative individuals with influenza-like illness (also identified from laboratory data). The sensitivity of the methods to confounders that were considered as relevant was tested with a simulation. Both methods were applied to the data of the German influenza surveillance data of the season 2004/2005. The estimated effectiveness over all age groups was rather low with both methods, but comparable with other estimations from the literature. We observed differences in certain age groups between the methods as well as large differences between particular age groups within one method. Possible explanations are random variations due to low numbers in age strata and other influences not yet considered. Therefore the estimations should be interpreted with care; however, relative comparisons among seasons may still be meaningful.

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