Year: 2012
Type: dissertation
Abstract: The family Picornaviridae includes many human pathogens. Human enteroviruses (HEVs) exhibit a variety of clinical manifestations ranging from poliomyelitis and encephalomyelitis to respiratory infections and rashes. Human rhinoviruses (HRVs) are the major causes of the common cold. Human parechoviruses (HPeVs) and Aichi virus (AV) are mostly detected in cases of gastroenteritis, and hepatitis A virus (HAV) causes hepatitis with favourable prognosis. In addition to HEVs and HRVs, a large number of viruses are recognized as respiratory pathogens. The conventional respiratory pathogens include influenza A and B viruses, human respiratory syncytial virus (RSV), adenoviruses (AdVs), parainfluenza viruses (PIVs) and the human coronaviruses (hCoVs) OC43 and 229E. Moreover, several new respiratory pathogens, such as human metapneumovirus (hMPV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the hCoVs HKU1 and NL63 have been found during the 2000s. Human bocavirus (hBoV) is also increasingly being recognized as a true pathogen of humans. Since many clinical illnesses may be caused by several different viruses, multiplex assays for simultaneous detection of several viruses are increasingly being applied. Real-time multiplex polymerase chain reaction (PCR) assays for detection of viral nucleic acids offer remarkable benefits, such as short turnaround time and the non-necessity for handling amplified products. Since multiplexing, utilizing real-time PCR, is limited by reduction in amplification efficiency due to multiple primer and probe sets, separate amplification and hybridization reactions have re-emerged in attempts to develop tests with broad diagnostic range. With this approach microarrays, which have the potential for resolving complex mixtures of amplification products, may be applied. In this study, a multiplex reverse transcription-PCR (RT-PCR) and liquid hybridization assay for sensitive detection of HEV, HRV, HPeV and AV were developed and a single RT-PCR and liquid hybridization assay for detection of HAV was optimized. In analysis of clinical samples, the results obtained by the multiplex assay were consistent with those obtained by routine diagnostic assays. When 68 stool samples were analysed for the presence of HPeV and AV, one sample positive for HPeV was detected. This finding is in line with the current knowledge of neither of these viruses being very common enteric pathogens. More rapid detection of HEV and HRV in respiratory samples was achieved when a real-time duplex RT-PCR assay for detection of these viruses was developed. The same approach was used to develop another assay for more sensitive detection of RSV than with the direct fluorescent assay (DFA) and simultaneous identification of hMPV. Both multiplex real-time RT-PCR assays provided reliable and sensitive detection of their targets, except for detection of HRV, since doubts were raised on the ability of the assay to detect all rhinoviruses. Moreover, two commercial hMPV antibodies were found applicable for detection of the virus in respiratory samples by DFA. Results from analysis of respiratory samples using the duplex real-time RT-PCR assays were compared with those obtained with DFA and the Respiratory Viral Panel (RVP) Fast test, a bead-based suspension microarray test evaluated for routine diagnosis. The RVP Fast assay and PCR showed similar detection rates, except for HEV/HRV, for which a higher detection rate by RVP was observed. All PCR-based assays presented more findings of their target viruses than DFA. The broad detection range of the RVP Fast assay resulted in a nearly threefold overall detection rate, compared with that by DFA. Moreover, analysis of clinical samples resulted in a notable prevalence of hMPV and non-SARS-hCoVs, which emphasizes the role of these viruses as respiratory pathogens. Although the RVP Fast assay demonstrated adequate overall performance, doubts were raised on the ability of the test to detect the H1N1 2009 influenza A virus and all AdV serotypes. Evaluation of the RVP Fast assay demonstrated the remarkable increase in overall viral detection rate that results from adapting a PCR-based multiplex assay to virus diagnostics. The sensitive detection of all the viruses of clinical relevance facilitates efficient infection control measures and appropriate patient management and enables systematic studies on the clinical importance of coinfections. Moreover, collection of data on occurrence of all the viruses of clinical relevance will enable a better understanding of the seasonality, geographical distribution and risk groups of the viral pathogens.
Source: Työväentutkimus Vuosikirja
Author: Pia Jokela
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1499029268