Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections

Abstract

For the detection of respiratory viruses conventional culture techniques are still considered as the gold standard. However, results are mostly available too late to have an impact on patient management. The latest developments include appropriate DNA- and RNA-based amplification techniques (both NASBA and PCR) for the detection of an extended number of agents responsible for LRTI. Real time amplification, the latest technical progress, produces, within a considerable shorter time, results with a lower risk of false positives. As results can be obtained within the same day, patient management with appropriate therapy or reduction of unnecessary antibiotic therapy in LRTI will be possible. A number of technical aspects of these amplification assays, and their advantages are discussed. The availability and use of these new diagnostic tools in virology has contributed to a better understanding of the role of respiratory viruses in LRTI. The increasing importance of the viral agents, Mycoplasma pneumoniae and Chlamydophila pneumoniae in ARI is illustrated. A great proportion of ARI are caused by viruses, but their relative importance depends on the spectrum of agents covered by the diagnostic techniques and on the populations studied, the geographical location and the season. The discovery of new viruses is ongoing; examples are the hMPV and the increasing number of coronaviruses. Indications for the use of these rapid techniques in different clinical situations are discussed. Depending on the possibilities, the laboratory could optimize its diagnostic strategy by applying a combination of immunofluorescence for the detection of RSV an IFL, and a combination of real-time amplification tests for other respiratory viruses and the atypical agents. When implementing a strategy, a compromise between sensitivity, clinical utility, turn around time and cost will have to be found.

Prevalence and Molecular Characterization of Circulating Respiratory Syncytial Virus (RSV) in Chennai, South India during 2011-2014

“…One fourth (2.5 millions) of the total deaths among children less than 5 years of age occur in India and approximately 20% of these are due to ARTIs [2][3][4] . Other than that bacterial infection, Influenza virus and Human respiratory syncytial virus (HRSV) have been identified as the predominant etiological agents for lower respiratory tract infections [5][6][7] . About 20-40 % of the children are hospitalized due to RSV with ARI in India [8][9][10] .…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…Nucleic acid amplification using thermostable polymerase (PCR) was initially reported in 1988, and this method remains largely unchanged as it forms the backbone of molecular diagnostics in clinical microbiology laboratories today (6). Properties such as high sensitivity and specificity, an extremely low limit of detection (1 to 10 copies of the target), and rapid results have led to proposed changes in the definition of the "gold standard" method for detection and identification of microorganisms in clinical specimens, especially for those that are difficult to culture, including fastidious bacterial or viral pathogens (7)(8)(9)(10). While the basic principle of nucleic acid amplification tests (NAATs) has not changed, technologies surrounding this core, including amplification strategy, amplicon detection, multiplexing of reactions, and automation of the entire process into sample-to-result platforms, have provided a large menu of options for the molecular microbiology laboratory to choose from.…”

“…Both require preextraction of nucleic acids to obtain template and manual pipetting of each PCR component or master mix into individual RT-PCR tubes. Multiplex assays using analyte-specific reagents (ASRs) for influenza viruses A and B, respiratory syncytial viruses (RSV) A and B, and HSV-1 and -2 have demonstrated high sensitivities compared to other rapid tests, and results are available days earlier than with viral culture methods (10,(69)(70)(71)(72). A recently developed and FDA-cleared test for the detection of bacterial causes of enteritis demonstrated 100% sensitivity and Ͼ99% specificity for 5 targets (Salmonella spp., Shigella spp., Campylobacter coli/jejuni, stx 1 , and stx 2 ) compared to culture and an alternative molecular assay (73).…”

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint

“…Also, target DNA has been obtained from different specimens, such as sputum, nasopharyngeal or pharyngeal swabs, brochoalveolar lavages or pleural fluid, and then comparisons of performance between these assays are difficult. For comprehensive understanding of the use of NATs for the detection of M. pneumoniae, genital mycoplasmas and other respiratory pathogens in clinical specimens, see the reviews done by Ieven, 2007;;Lo & Kam, 2006;Loens et al, 2003bLoens et al, , 2010 As with any other diagnostic test, PCR assays designed for mycoplasma detection in the clinical setting offer several advantages over other non-molecular tests, but still have several drawbacks to take into account (Table 3). Notwithstanding, there are several primer sets that have been successfully applied for diagnosis of mycoplasmal diseases in humans (Table 4).…”