Evaluation of a smartphonebased rapid fluorescent

Authors: SeonJu Yeo Bui Thi Cuc Haan Woo Sung Hyun Park

Publish Date: 2016/06/10

Volume: 161, Issue: 8, Pages: 2249-2256

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Abstract

Repeated interspecies transmission of H9N2 virus from poultry to humans and human infections transmitted via aerosols highlight the need for a highly sensitive rapid diagnostic system for the detection of this virus However no such test exhibiting high performance has been developed In this study the performance of a smartphonebased rapid fluorescent diagnostic system SRFDS was optimized for the diagnosis of an H9N2virusinfected animal To suppress the nonspecific reactivity of the bioconjugate in oropharyngeal OP and cloacal CL samples derived from chickens different blocking reagents were tested and a mixture of casein and sucrose was found to be optimal To assess the performance of SRFDS OP and CL samples were obtained from specificpathogenfree chickens and used for comparison of this method with realtime reverse transcription PCR rRTPCR at time points of three five and seven days postinfection dpi The limit of detection of SRFDS was found to be 75 PFU/mL which was 138fold higher than that of a conventional colloidalgoldbased avian influenza rapid diagnostic test In the animal study the presence of viral antigen was monitored with SRFDS and the relative sensitivity relative to rRTPCR results was 9444  17/18 and 9523  20/21 in OP and CL specimens respectively The specificity of SRFDS was 100  These results imply that the diagnostic performance of SRFDS might be comparable to that of rRTPCR for diagnosis of H9N2 in chickens and that this test can be used as a highly sensitive rapid diagnostic method in field studies on broiler poultry and wild birdsAlthough H9N2 avian influenza viruses generally cause only mild to moderate disease in coinfections with other viruses and bacteria approximately 70  morbidity and 30  mortality have been reported in poultry 1 2 In contrast to most avian influenza viruses that have a preference for alpha 23linked sialic acid SA receptors some H9N2 viruses are able to recognize alpha 26linked SA receptors for direct transmission to humans 3 This raises the fear that they may become pandemic through repeated interspecies transmission from poultry to humans Moreover as aerosol transmission of H9N2 infection has been reported timely surveillance of H9N2 is essential 4To improve surveillance an efficient and accurate rapid diagnostic method to detect H9N2 viruses in both poultry and humans is indispensable for pandemic preparedness Studies on influenza virus shedding are important to understand the epidemiology of the virus and they also form the basis for rational diagnostic strategies 5 An animal model of influenza has been used to understand viral and host factors that contribute to transmission outcomes but so far few trials have been performed to improve rapid diagnostic tests RDTs As animal models are living specimens the relationship between the amounts of viral RNA and viral antigen is biologically relevant and thus can validate the quality and accuracy of a rapid diagnostic systemCurrently rapid diagnostic tests RDTs vary in their sensitivity and specificity when compared to RTPCR According to CDC guidelines upper respiratory samples should be used for influenza virus RDT In addition the use of RDTs in hospitalized patients is not encouraged where RTPCR is available because the sensitivity is approximately 50–70  and the specificity is approximately 90–95  6To improve the accuracy and sensitivity of RDTs many recent trials have employed fluorescent technology within this platform 7 8 9 Previously we developed a smartphonebased rapid fluorescent diagnostic system SRFDS with fluorescent coumarinderived dendrimerbased bioconjugation and lightemitting diode LED modules to detect H5N1 virus in human throat samples 9 However the performance of SRFDS in the diagnosis of poultry was unclearVarious specimens such as respiratory tract specimens and fecal specimens need to be tested using a highperformance RDT because after the primary respiratory infection H9N2 virus multiplies in the intestinal tract of chickens and is transmitted through feces 10 11 In humans detection of influenza virus RNA and viable influenza virus in stool suggests that influenza virus can be localized to the gastrointestinal tract of children and this could serve as a mode of transmission during seasonal and epidemic influenza outbreaks 12 In severe cases stool specimens have been subjected to rRTPCR and virus isolation targeting the influenza RNA matrix M gene 13 Therefore a highly sensitive rapid diagnostic system for fecal samples is essential for efficient identification and management of influenza cases in poultry and humans

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