Emerging Advances in Rapid Diagnostics of Respiratory Infections David R. Murdoch*, Lance C. Jennings, Niranjan Bhat, Trevor P. Anderson Department of Pathology, University of Otago Christchurch, PO Box 4345, Christchurch 8140, New Zealand Microbiology Unit, Canterbury Health Laboratories, PO Box 151, Christchurch 8140, New Zealand Division of Infectious Diseases, Department of Pediatrics, Johns Hopkins School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA

Diagnostic laboratories  central role in the recognition of new and emerging infections ▫SARS coronavirus, 2003 ▫Novel H1N1 influenza A strain, 2009 Nonviral respiratory pathogens ▫Less profound developments in lab technology ▫Modest improvements Microscopy and culture of respiratory specimens, blood cultures, detection of antigens in urine and upper respiratory specimens, and serology ⇒ antigen and nucleic acid detection

Several major challenges hindering the search for the causes of respiratory infections ▫Difficulty collecting lower respiratory specimens ▫Problems distinguishing colonization from infection ▫Poor clinical (diagnostic) sensitivity of assays ▫Inadequate evaluation of new diagnostics

Antigen Detection Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), latex agglutination, coagulation, and chromatographic immunoassay Limited range of pathogens ▫Detection of selected bacterial pathogens in urine ▫Detection of viruses in respiratory specimens Streptococcus pneumoniae ▫Newer generation immunochromatographic test – C- polysaccharide cell wall antigen in urine (NOW)  Sensitivity of 70-80%, specificity of greater than 90%  In children, not reliable for pneumococcal carriage ▫Pneumolysin ▫Combination of pneumolysin-specific antigen detection ELISA with NOW test  better diagnostic yield

Legionella pneumophila ▫Detection of soluble Legionella antigen in urine  Only reliably detect infection caused by L. pneumophila serogroup 1 Detection of respiratory viral antigens in respiratory secretions ▫Influenza viruses, respiratory syncytial virus (RSV), parainfluenza viruses, adenoviruses, and human metapneumovirus ▫Technical expertise ▫Advantage of allowing direct evaluation of specimen quality ▫Commercial rapid diagnostic tests (RDTs)  Influenza or RSV  Dipsticks, cassettes, or cards  5-40 minutes

Nucleic Acid Amplification Tests Relevance of new agents such as human metapneumovirus New insights into previously recognized ones such as rhinoviruses Advantages over more traditional techniques ▫Improved sensitivity ▫Rapid genetic information regarding sequence evolution, geographic variation, or the presence of virulence factors or antibiotic resistance ▫Rapid turn-around time ▫Test for multiple pathogens simultaneously ▫Automation ▫Lower safety risk than culture

PCR ▫Most common and thoroughly evaluated method ▫Conventional, real-time, and multiplex platforms ▫Various amplicon detection methods such as gel analysis, ELISA, DNA hybridization, or the use of fluorescent dyes or chemical tags ▫Real-time PCR  2 steps of amplification and detection combined in 1 step  increased speed and efficiency of testing, reduced risk of operator error and cross-contamination  Possibility of quantifying the amount of starting nucleic acid material

▫Multiplex PCR  Multiple PCR targets sought after simultaneously in one reaction  Wider acceptance  Advantages of increasing the number of pathogens tested for, without increasing the required amount of operator time or specimen material  Amplification step  Nested primer combinations, complex primer structures, and nontraditional nucleotides

 Detection stage  Solid-phase arrays, such as polystyrene microbead suspensions that use fluorescent dyes to differentiate targets, or the microchip formats that identify targets by binding to a specific physical location ▫micro-bead suspensions – targets ▫Microchip formats – few dozen to thousands of targets  Microarrays ▫Increased breadth of targets ▫Decreased sensitivity  Agarose gel electrophoresis  Mass spectrometry ▫Good primer design  Bacteria – house keeping genes  Virus – limited size of viral genome  limited targets

Abbreviations: DFA, direct fluorescence assay; ND, not determined; TCID50, tissue culture infective dose needed to produce 50% change.

Abbreviations: ORF, open reading frame; UTR, untranslated region.

Published literature on NAATs can be difficult to interpret ▫Various study design ▫Rare head-to-head comparisons ▫Calculation of clinical sensitivity and specificity is complicated ▫Several NAATs require investment in specialized equipment  high-volume testing ▫Comparison of study results are problematic Few NAAT assays for respiratory diagnosis are licensed for clinical use

NAATs for specific respiratory pathogens Respiratory viruses ▫Most sensitive diagnostic approach ▫Current “gold standards” (culture and direct immunofluorescence) will be eventually replaced by NAATs ▫PCR  Diagnostic test of choice for some respiratory infections  Useful epidemiologic tool for characterizing the role of viruses in various disease states Pneumococcal disease ▫PCR  Sensitivity of % in blood sample, higher sensitivity in children  Sputum samples from adults with pneumonia, positive in % of the patients

 Further refinement  Multiple targets  increased specificity  With lytA assays – advantages over other assays  Quantification of S. pneumoniae DNA load  Distinguish colonization from infection ▫Higher bacterial burden in pneumococcal disease than in carrier state  Higher pneumococcal DNA loads – severe disease

Detection of respiratory pathogens difficult to culture ▫Mycoplasma pneumoniae  Extensive evaluation of 13 antibody detection assays  Few commercial assays – sufficient sensitivity & specificity  Throat swabs and nasopharyngeal samples – high sensitivity, specificity and convenience ▫Legionnaires’ disease  Legionella spp.  PCR – sensitivity ≥ culture (lower respiratory specimens)  Nonrespiratory specimens such as urine, serum, & pph WBC ▫Chlamydophila pneumoniae  Few evaluations  Great variety in the methods ▫Bordetella pertussis  PCR – positive for a longer period after the onset of symptoms

▫Pneumocystis jiroveci  PCR- greater sensitivity than cytologic methods  Colonization of uncertain clinical significance  PCR (+), standard methods (-) ▫Tuberculosis  NAATs for mycobacteria – failing to provide greater sensitivity than culture-based methods  Relatively high false-negative rate with NAATs  Paucibacillary nature of samples  Presence of inhibitors in samples  Suboptimal DNA extraction methods  High specificity (>98%), variable senstivity (90-100% for smear positive, % for smear negative)  Detection of mycobacterial DNA in urine, direct detection in respiratory specimens by microassays

New pathogen discovery Microassays High-throughput sequencing proteomics

Breath Analysis Alveolar breath ▫Many biomarkers derived from the blood by passive diffusion across the alveolar membrane and direct markers of lung injury Breath testing ▫Noninvasive, easily repeatable, and minimal specimen workup Electric nose devices ▫Pneumonia in mechanically ventilated patients Microorganisms – volatile metabolites that may be used as biomarkers ▫Gaschromatography/mass spectroscopy ▫Difficult to discover unique markers for each pathogens produced in sufficient quantities to enable detection ▫Aspergillus fumigatus and M. tuberculosis

Further Prospects Antigen-detection assays in immunochromatographic or similar formats ▫Rapid, simple to perform ▫Near-patients tests NAATs ▫Multiplex assays that detect the common respiratory pathogens New approaches ▫Breath analysis