Dr. Markotić indicated variable levels of satisfaction with POC tests. When she initially tried using POC tests to detect Puumala and Dobrava hantaviruses, she experienced too many false positives; recently, however, the quality of commercial tests has improved and she has resumed using them. She also uses POC tests to look for influenza A, Dengue fever virus, and Chikungunya. Dr. Markotić emphasized that while POC tests in conjunction with a clinical evaluation can serve as a valuable early indicator, she always proceeds with additional confirmatory testing.

Dr. Markotić also compared the costs of various techniques. For example, given the short shelf life of many ELISA reagents and the frequency with which she does the tests for clinical work, she indicated that PCR tests are comparatively cheaper for her lab. She reported mainly using ELISA tests for research projects when she knows the reagents will be used before they expire. Although University Hospital for Infectious Diseases has in-house sequencing capabilities, she normally uses the equipment at the Institute for Biological Services, which is cheaper. She noted, however, that the cheapest option is to send the samples to South Korea. Dr. Markotić pointed out that, in general, costs in Croatia are extremely high, and reagents often cost 5-6 times more than they would in the United States.

She explained that her research uses a number of culture-based techniques that are performed in BSL-3 conditions and that she would like to pursue more immunopathogenesis research. In particular, she developed a research model for hantaviruses using 293-HEK cells (Markotic et al., 2003). Resource limitations have, however, put this research on hold. Her lab, however, is currently working to develop molecular tests for Crimean-Congo hemorrhagic fever (CCHF), other hemorrhagic fevers, poxvirus, and rickettsia. As the Croatian Ministry of Health has limited opportunities that support research, Dr. Markotić indicated that she relies on international collaborations and foreign research grants for support.

Biorisks Connected with Wild Birds: Results of Avian Influenza Virus Surveillance in Southwest Siberia (Russia) in 2010

Alexander Shestopalov (State Research Center of Virology and Biotechnology, Russia), whose division monitors influenza in humans, wild birds, and poultry and contributes their data to WHO, described his lab’s efforts to understand the dynamics of highly pathogenic avian influenza (HPAI) in Eurasia.

Dr. Shestopalov started by explaining that southwest Siberia contains many lakes, three major flyways for migrating birds, and played an important role in the 2005 expansion of influenza A, H5N1 in Eurasia. He believes that the lakes and rivers in the region play an important role in the circulation of avian influenza (AI) and that by monitoring influenza activity in migratory and resident bird there, they could provide an early warning system for HPAI outbreaks in birds in Eurasia.

In 2010, his lab isolated 32 AI viruses from 743 samples collected in western Siberia. Phylogenetic subtyping using PCR and sequencing indicated that 10 were H3N8, 4 were H3N6, and 8 were H4N6, while 10 could not be typed.

Another of the lab’s datasets demonstrated that the H5N1 viruses present on Uvs–Nuur Lake in Mongolia near the Russian border shifted from clade 2.2 to clade 2.3.2 (evolutionarily distinct groups) between 2006 and 2009-2010. Hemagglutination inhibition assays using sera from ferrets showed no cross-reactivity between either virus and the opposite sera, suggesting that the viruses produce distinct immunological responses and display distinct antigens. As such, Dr. Shestopalov proposed that the current circulation of clade 2.3.2 and the disappearance of clade 2.2 could be explained by antigenic drift of the hemagglutinin under the pressure of population immunity in the natural host species. Clade 2.3.2 viruses were subsequently found in Russian in 2009, Bulgaria and Romania in 2010, and Japan in 2011, illustrating the potential for the region to function as an early warning system.

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