Download Emerging Advances in Rapid Diagnostics of Respiratory Infections

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
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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 – Cpolysaccharide 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 – 17-20 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 29-100% in blood sample, higher sensitivity in
children
 Sputum samples from adults with pneumonia, positive in 68100% 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, 33-100% 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